ES2707499T3 - Pneumococcal polysaccharide conjugate vaccine - Google Patents

Pneumococcal polysaccharide conjugate vaccine Download PDF

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ES2707499T3
ES2707499T3 ES15195398T ES15195398T ES2707499T3 ES 2707499 T3 ES2707499 T3 ES 2707499T3 ES 15195398 T ES15195398 T ES 15195398T ES 15195398 T ES15195398 T ES 15195398T ES 2707499 T3 ES2707499 T3 ES 2707499T3
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protein
immunogenic composition
saccharide
vaccine
paragraph
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Ralph Leon Biemans
Nathalie Marie-Josephe Garcon
Philippe Vincent Hermand
Jan Poolman
Mechelen Marcelle Paulette Van
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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Priority to GB0526232A priority Critical patent/GB0526232D0/en
Priority to GBGB0607088.2A priority patent/GB0607088D0/en
Priority to GB0607087A priority patent/GB0607087D0/en
Priority to GB0609902A priority patent/GB0609902D0/en
Priority to GB0620337A priority patent/GB0620337D0/en
Priority to GBGB0620336.8A priority patent/GB0620336D0/en
Priority to GB0620815A priority patent/GB0620815D0/en
Priority to GB0620816A priority patent/GB0620816D0/en
Priority to PCT/GB2006/004634 priority patent/WO2007068907A2/en
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    • AHUMAN NECESSITIES
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    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/385Haptens or antigens, bound to carriers
    • AHUMAN NECESSITIES
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    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092Streptococcus
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    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
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    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/545Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/55Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • AHUMAN NECESSITIES
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55566Emulsions, e.g. Freund's adjuvant, MF59
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    • A61K2039/575Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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    • A61K2039/60Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031Proteins
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    • A61K2039/60Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031Proteins
    • A61K2039/6037Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00Medicinal preparations containing antigens or antibodies
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    • A61K2039/6031Proteins
    • A61K2039/6068Other bacterial proteins, e.g. OMP
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    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/60Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087Polysaccharides; Lipopolysaccharides [LPS]
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    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/62Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/70Multivalent vaccine
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/38Medical treatment of vector-borne diseases characterised by the agent
    • Y02A50/408Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a protozoa
    • Y02A50/411Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a protozoa of the genus Plasmodium, i.e. Malaria
    • Y02A50/412Medical treatment of vector-borne diseases characterised by the agent the vector-borne disease being caused by a protozoa of the genus Plasmodium, i.e. Malaria the medicinal preparation containing antigens or antibodies, e.g. vaccines, antisera

Abstract

An immunogenic composition comprising conjugates of S. pneumoniae capsular saccharides of serotypes 19A and 19F in which 19A is conjugated with a first bacterial toxoid that is pneumolysin, diphtheria toxoid or CRM197 and 19F is conjugated with a second bacterial toxoid that is toxoid diphtheria or CRM197 and further comprising conjugates of the capsular saccharides of S. pneumoniae 4, 6B, 9V, 14, 18C, 23F, 1, 5 and 7F, in which the average size of saccharide 19A is between 110 and 700 kDa.

Description

DESCRIPTION

Pneumococcal polysaccharide conjugate vaccine

Field of the invention

The present invention relates to an improved Streptococcus pneumoniae vaccine.

BACKGROUND OF THE INVENTION

Children under 2 years of age do not develop an immune response against most polysaccharide vaccines, so it has been necessary to make polysaccharides immunogenic by chemical conjugation with a protein carrier. Coupling of the polysaccharide, a T-independent antigen, to a protein, a T-dependent antigen, confers to the polysaccharide the T-dependency properties that include an isotope change, affinity maturation, and memory induction.

However, there may be problems with the repeated administration of protein-polysaccharide conjugates, or the combination of protein-polysaccharide conjugates to form multivalent vaccines. For example, a polysaccharide vaccine (PRP) of Haemophilus influenzae type b using tetanus toxoid (TT) as a protein carrier has been reported to be tested in a dosing interval with simultaneous immunization with TT (free) and a polysaccharide conjugate vaccine. pneumococcal-TT following a conventional childhood immunization schedule. Upon increasing the dosage of the pneumococcal vaccine, the immune response to the PRP polysaccharide portion of the Hib conjugate vaccine decreased, indicating an immune interference of the polysaccharide, possibly through the use of the same protein carrier (Dagan et al., Infect Immun. (1998); 66: 2093-2098).

The effect of the dosage of the protein carrier on the humoral response to the protein itself has also been proven to be multifaceted. In human children it has been reported that the increase of a tetravalent tetanus toxoid conjugate resulted in a decrease in the response to the tetanus vehicle (Dagan et al., Supra). Classical analyzes of these effects of the vaccine combination have been described as vehicle-induced epitope suppression, which is not fully understood but which is believed to result from an excessive amount of protein carrier (Fattom, Vaccine 17: 126 (1999)). ). This seems to result in competition with Th lymphocytes, by B lymphocytes against the protein carrier, and B lymphocytes against the polysaccharide. If the B lymphocytes against the protein carrier predominate, there are not enough Th lymphocytes available to provide the necessary help for the polysaccharide-specific B lymphocytes. However, the immunological effects have not been consistent, increasing in some cases the immune response with the total amount of protein carrier, and in other cases decreasing the immune response.

Therefore, technical difficulties remain in the combination of multiple polysaccharide conjugates in a single effective vaccine formulation.

Streptococcus pneumoniae is a Gram-positive bacterium responsible for considerable morbidity and mortality (particularly in young and old), which produces invasive diseases such as pneumonia, bacteremia and meningitis, and diseases associated with colonization, such as acute otitis media. The rate of pneumococcal pneumonia in the USA UU for people over 60 years of age it is estimated that it is 3 to 8 per 100,000. In 20% of cases it leads to bacteremia, and other manifestations such as meningitis, with a mortality rate close to 30% even with antibiotic treatment.

The pneumococcus is encapsulated with a chemically bound polysaccharide that confers the serotype specificity. There are 90 known serotypes of pneumococci, and the capsule is the main determinant of virulence for pneumococci, since the capsule not only protects the internal surface of the complement bacteria, but it is also poorly immunogenic. Polysaccharides are antigens independent of T, and can not be processed or presented in MHC molecules to interact with T lymphocytes. However, they can stimulate the immune system through an alternative mechanism that involves cross-linking with surface receptors. of B lymphocytes

It has been shown in several experiments that protection against invasive pneumococcal disease corresponds very strongly with capsule-specific antibodies, and protection is serotype specific.

Streptococcus pneumoniae is the most common cause of invasive bacterial disease and otitis media in infants and young children. Likewise, the elderly develop poor responses to pneumococcal vaccines [Roghmann et al., (1987), J. Gerontol. 42: 265-270], therefore, the incidence of bacterial pneumonia increases in this population [Verghese and Berk, (1983) Medicine (Baltimore) 62: 271-285].

WO03 / 051392 discloses a vaccine conjugate of multiple serotypes of Streptococcus pneumoniae.

It is an object of the present invention to develop an improved formulation of a multi-serotype polysaccharide vaccine conjugate.

Brief description of the Figures

Figure 1. Bar graph showing the immunogenicity of an 11-valent conjugate in elderly Rhesus monkeys. The lightest bars represent the GMC after two inoculations with the 11-valent conjugate in an aluminum phosphate adjuvant. The darker bars represent the GMC after two inoculations with the 11-valent conjugate in adjuvant C.

Figure 2. Bar graph showing the memory B cells for PS3 after inoculation of the 11-valent conjugate in adjuvant C or aluminum phosphate adjuvant.

Figure 3. Bar graph showing the immunogenicity against the 19F polysaccharide in Balb / c mice for the flat 4-valent polysaccharides and the 4-valent dPly conjugates.

Figure 4. Bar graph showing the immunogenicity against the 22F polysaccharide in Balb / c mice for the flat 4-valent polysaccharides and the 4-valent PhtD conjugates

Figure 5. Bar graph showing the anti-22F IgG response in Balb / c mice.

Figure 6. Bar graph showing the titles of opsono-phagocytosis against 22F in Balb / c mice.

Figure 7. Bar graph comparing the IgG response induced by young C57B1 mice after immunization with the 13-valent vaccine conjugate formulated with different adjuvants.

Figure 8. Bar chart showing the protection efficacy of different vaccine combinations in a pneumonia model in monkeys.

Figure 9. Bar graph showing the anti-PhtD IgG response in Balb / c mice after immunization with 22F-PhtD or 22F-AH-PhtD conjugates.

Figure 10. Protection against a type 4 pneumococcal challenge in mice after immunization with 22F-PhtD or 22F-AH-PhtD.

Description of the invention

The present invention provides an immunogenic composition comprising conjugates of capsular saccharide Streptococcus pneumoniae serotypes 19A and 19F in which 19A is conjugated with a first bacterial toxoid, which is pneumolysin, diphtheria toxoid or CRM197 and 19F is conjugated with a second toxoid is diphtheria toxoid or CRM197 and further comprises conjugates of the capsular saccharides of S. pneumoniae 4, 6B, 9V, 14, 18C, 23F, 1.5 and 7f, in which the average size of saccharide 19A is above 100 kDa

The term "capsular saccharide" includes capsular polysaccharides and oligosaccharides that can be derived from capsular polysaccharides. An oligosaccharide contains at least 4 sugar moieties. The terms "conjugate" and "conjugate" refer to a capsular saccharide covalently linked to a protein carrier.

For the purposes of the present invention, "immunizing a human host against COPD exacerbations" or "treatment or prevention of COPD exacerbations" or "reducing the severity of COPD exacerbations" refers to a reduction in the incidence or rate of COPD exacerbations. of COPD exacerbations (for example, a reduction in the rate of 0.1, 0.5, 1, 2, 5, 10, 20% or more), for example in a group of patients immunized with the compositions or vaccines of the invention.

The term bacterial toxoid includes bacterial toxins that have been inactivated by genetic mutation, by chemical treatment or by conjugation. Suitable bacterial toxoids include tetanus toxoid, pertussis toxoid, bacterial cytolysins or pneumolysin. Pneumolysin (Ply) mutations that decrease the toxicity of pneumolysin have been described (WO 90/06951, WO 99/03884). Similarly, genetic mutations of diphtheria toxin that decrease its toxicity are known (see below). Genetically detoxified diphtheria toxin analogs include CRM197 and other mutants described in US 4,709,017. US 5,843,711. US 5,601,827, and US 5,917,017. CRM197 is a non-toxic form of diphtheria toxin but does not distinguish immunologically from diphtheria toxin. CRM197 is produced by C. diphtheriae infected by the non-toxigenic p197tox phase created by nitrosoguanidine mutagenesis of the toxigenic b-carinophagus (Uchida et al., Nature New Biology (1971) 233; 8-11). CRM197 protein has the same molecular weight as diphtheria toxin but differs from it by a single base change in the structural gene. This results in a change from a glycine to a glutamine at the amino acid position 52 which makes a fragment A incapable of binding to NAD and therefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem 46; 69-94, Rappuoli Applied and Environmental Microbiology Sept 1983 p560-564).

The first and second bacterial toxoids can be the same or different. When the first and second bacterial toxoids are different, it means that they have a different amino acid sequence.

For example, 19A and 19F can be conjugated to diphtheria toxoid and diphtheria toxoid; CRM197 and CRM197; toxoid diphtheria and CRM197, CRM197 and diphtheria toxoid; pneumolysin and diphtheria toxoid; or pneumolysin and CRM197, respectively.

In one embodiment, in addition to conjugates of S. pneumoniae saccharides of 19A and 19F, the immunogenic composition further comprises conjugates of capsular saccharides of S. pneumoniae 1, 4, 5, 6B, 7F, 9V, 14, 18C, 22F and 23F .

In one embodiment, in addition to the saccharide conjugates of S. pneumoniae 19A and 19F, the immunogenic composition comprises conjugates of capsular saccharides of S. pneumoniae 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 22F and 23F.

In one embodiment, in addition to the saccharide conjugates of S. pneumoniae of 19A and 19F, the immunogenic composition comprises conjugates of capsular saccharides of S. pneumoniae 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C , 22F and 23F.

Normally the Streptococcus pneumoniae vaccine of the present invention will comprise saccharide capsular antigens (optionally conjugated), wherein the saccharides are derived from at least ten serotypes of S. pneumoniae. The number of capsular saccharides of S. pneumoniae can vary from at least 10 different serotypes (or "V", by valence) to 23 different serotypes (23V). In one embodiment there are 11, 12, 13, 14 or 15 different serotypes. In another embodiment of the invention, the vaccine may comprise S. pneumoniae saccharides and non-conjugated saccharides of S. pneumoniae. Optionally, the total number of saccharide serotypes is less than or equal to 23. The vaccine may comprise 11, 12, 13, 14 or 16 conjugated saccharides and 12, 11, 10, 9 or 7 respectively, unconjugated saccharides.

In one embodiment the multivalent pneumococcal vaccine of the invention will be selected from the following serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F , 18C, 19A, 19F, 20, 22F, 23F and 33F, although it is appreciated that one or two different serotypes could be substituted depending on the age of the recipient receiving the vaccine and the geographical location in which the vaccine will be administered. For example, a 10-valent vaccine may comprise the polysaccharides of serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valent vaccine may also include serotype 3 serotypes. A 12- or 13-valent pediatric (baby) vaccine may also include the 11-valent formulation supplemented with serotypes 6A and 19A, or 6A and 22F, or 19A and 22F, or 6A and 15B, or 19A and 15B, or 22F and 15B, while a vaccine for 13-valent elderly may include the 10 or 11-valent formulation supplemented with serotypes 19A and 22F, 8 and 12F, or 8 and 15B, or 8 and 19A, or 8 and 22F, or 12F and 15B, or 12F and 19A, or 12F and 22F, or 15B and 19A, or 15B and 22F. a 14-valent pediatric vaccine may include the 10-valent formulation described above supplemented with serotypes 3, 6A, 19A and 22F; serotypes 6A, 8, 19A and 22F; serotypes 6A, 12F, 19A and 22F; serotypes 6A, 15B, 19A and 22F; serotypes 3, 8, 19A and 22F; serotypes 3, 12F, 19A and 22F; serotypes 3, 15B, 19A and 22F; serotypes 3, 6A, 8 and 22F; serotypes 3, 6A, 12F and 22F; or serotypes 3, 6A, 15B and 22F.

In a further embodiment of the invention, at least 2 or 13 antigenic saccharides are included, for example a vaccine may comprise capsular saccharides derived from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F or capsular saccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F, although additional antigenic saccharides are also contemplated, for example 23 valences (such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F).

The vaccine of the present invention may comprise protein D (PD) from Haemophilus influenzae (see, for example, EP 0594610). Haemophilus influenzae is a key causative organism of otitis media, and the present inventors have shown that including this protein in a Streptococcus pneumoniae vaccine will provide a level of protection against otitis media related to Haemophilus influenzae (reference to POET publication). In one embodiment, the vaccine composition comprises protein D. In one aspect, PD is present as a protein carrier for one or more of the saccharides. In another aspect, protein D could be present in the vaccine composition as a free protein. In a further aspect, protein D is present as a protein carrier and as a free protein. Protein D can be used as a full-length protein or as a fragment (WO0056360). In a further aspect, protein D is present as a protein carrier for most saccharides, for example 6, 7, 8, 9 or more of the saccharides can be conjugated to protein D. In this aspect, protein D it can also be present as a free protein.

The vaccine of the present invention comprises one, two, or more different types of protein carriers. Each type of protein carrier can act as a vehicle for more than one saccharide, whose saccharides can be the same or different. For example, serotypes 3 and 4 can be conjugated with the same protein carrier, either with the same protein carrier molecule or with different molecules of the same protein carrier. In one embodiment, two or more different saccharides can be conjugated, either to the same protein carrier molecule or to different molecules of the same protein carrier.

Any of the capsular saccharides of Streptococcus pneumoniae present in the immunogenic composition of the invention other than 19A and 19F can be conjugated with a protein carrier independently selected from the group consisting of TT, DT, CRM197, TT C fragment, PhtD, PhtBE, or PhtDE (particularly those described in WO 01/98334 and WO 03/54007), detoxified pneumolysin and protein D. A more complete list of protein carriers that can be used in the conjugates of the invention is presented below.

The protein carrier conjugated to one or more of the capsular saccharides of S. pneumoniae in the conjugates present in the immunogenic compositions of the invention is optionally a member of the family of polyhistidine (Pht) triad proteins, fragments or fusion proteins. thereof. The PhtA, PhtB, PhtD or PhtE proteins may have an amino acid sequence that shares 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with a sequence disclosed in WO documents 00/37105 or WO 00/39299 (for example, with amino acid sequence 1-838 or 21-838 of SEQ ID NO: 4 of WO 00/37105 for PhtD). For example, the fusion proteins are composed of full length or 2, 3 or 4 fragments of PhtA, PhtB, PhtD, PhtE. Examples of fusion proteins are PhtA / B, PhtA / D, PhtA / E, PhtB / A, PhtB / D, PhtB / E. PhtD / A. PhtD / B, PhtD / E, PhtE / A, PhtE / B and PhtE / D, in which the proteins are linked with the first mentioned at the N-terminus (see, for example, WO01 / 98334).

When the Pht protein fragments are used (separately or as part of a fusion protein), each fragment optionally contains one or more histidine triad motifs and / or supercoiled regions of said polypeptides. A triad motif of histidine is the polypeptide part having the sequence HxxHxH where H is histidine and x is an amino acid other than histidine. A supercoiled region is a region provided by the "Coils" algorithm of Lupus, A et al., (1991) Science 252; 1162-1164. In one embodiment the or each fragment includes one or more histidine triad motifs as well as at least one supercoiled region. In one embodiment, the or each fragment contains exactly or at least 2, 3, 4, or 5 triad motifs of histidine (optionally, with the native Pht sequence between the 2 or more triads, or intra-triad sequence which is more 50, 60, 70, 80, 90 or 100% identical to a native pneumococcal intra-triad Pht sequence - for example, the intra-triad sequences shown in SEQ ID NO: 4 of WO 00/37105 for PhtD ). In one embodiment, the or each fragment contains exactly or at least 2, 3, or 4 supercoiled regions. In one embodiment a Pht protein disclosed herein includes the full-length protein with the bound signal sequence, the full-length protein matures with the signal peptide (eg, 20 amino acids at the N-terminus) removed, the naturally occurring variants of the Pht protein and immunogenic fragments of the Pht protein (eg, the fragments described above or polypeptides comprising at least 15 or 20 contiguous amino acids of an amino acid sequence of WO 00/37105 or WO 00/39299 in which said polypeptide is capable of producing a specific immune response for said amino acid sequence of WO 00/37105 or Wo 00/39299).

In particular, the term "PhtD" as used herein includes the full-length protein with the linked signal sequence, the full-length protein matures with the deleted signal peptide (e.g., 20 amino acids from the N-terminus), PhtD variants of natural origin and immunogenic fragments of PhtD (for example, fragments as described above or polypeptides comprising at least 15 or 20 contiguous amino acids of an amino acid sequence of WO 00/37105 or WO 00/39299 in which said polypeptide is capable of producing a specific immune response for said amino acid sequence of WO 00/37105 or WO 00/39299 (for example, SEQ ID NO: 4 of WO 00/37105 for PhtD).

If the protein carrier is the same for 2 or more saccharides of the composition, the saccharides could be conjugated with the same molecule of the protein carrier (vehicle molecules having 2 or more different saccharides conjugated to them) [see for example the document WO 04/083251]. Alternatively, the saccharides can each be conjugated separately to different molecules of the protein carrier (each protein carrier molecule only has one type of saccharide conjugated thereto).

Examples of protein carriers that can be used in the present invention are DT (diphtheria toxoid), TT (tetanus toxoid) or RR fragment C, DT CRM197 (a mutant of DT), other point mutants of DT, such as CRM176, CRM228 , CRM 45 (Uchida et al., J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 in Asp, Gln or Ser and / or Ala 158 by Gly and other mutations disclosed in US 4709017 or US 4950740; or the fragment disclosed in US 5843711, pneumococcal pneumolysin (Kuo et al., (1995) Infect Immun 63; 2706-13) including Ply detoxified in some way, for example, dPLY-GMBS (WO 04081515, PCT / EP2005 / 010258) or dPLY-formalin, PhtX, which include PhtA, PhtB, PhtD, PhtE and fusions of Pht proteins, for example, PhtDE fusions, PhtBE fusions (WO 01/98334 and WO 03/54007), ( describes Pht AE in more detail below), OMPC (meningococcal outer membrane protein - usually extracted from serogroup B of N. meningitidis - EP0372501), PorB (from N. meningitidis), PD (protein D from Haemophilus inHuenzae - see example, EP 0 594 610 B), or immunologically functional equivalents thereof, synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93/17712, W or 94/03208), pertussis proteins (documents WO 98/58668, EP0471177), cytokines, lymphokines, fac Growth hormones or hormones (WO 91/01146), artificial proteins comprising multiple epitopes of human CD4 + T lymphocytes of antigens derived from different pathogens (Falugi et al., (2001) Eur J Immunol 31; 3816-3824) such as the N19 protein (Baraldoi et al., (2004) Infect Immun 72; 4884-7), pneumococcal surface PspA protein (WO 02/091998), iron uptake proteins (WO 01 / 72337), toxin A or B of C. difficile (WO 00/61761).

Nurkka et al., Pediatric Infectious Disease Journal. 23 (11): 1008-14, Nov 2004. They described an 11-valent pneumococcal vaccine with all serotypes conjugated to PD. However, the present inventors have shown that the opsono-phagocytosis activity was improved by antibodies induced with conjugates having 19F conjugated with DT compared to 19F conjugated with PD. In addition, the present inventors have shown that a greater cross-reactivity with 19A is observed when 19F is conjugated with DT. Therefore, a feature of the composition of the present invention is that serotype 19F is conjugated with DT or CRM197. In one aspect, serotype 19F is conjugated with DT. It is also a feature of the invention that serotype 19A is conjugated with pneumolysin, DT or CRM197. The remaining saccharide serotypes of the immunogenic composition can all be conjugated to one or more protein carriers other than DT (ie, only 19F is conjugated to DT), or they can be divided among one or more protein carriers other than DT and the same DT. In one embodiment, 19F is conjugated with DT or CRM197 and all remaining serotypes are conjugated with PD. In a further embodiment, 19F is conjugated with DT or CRM 197, and the remaining serotypes are divided between PD, and TT or DT or CRM197. In a further embodiment, 19F is conjugated with DT or CRM197 and no more than one saccharide is conjugated with TT. In one aspect of this embodiment, said one saccharide is 18C or 12F. In a further embodiment, 19F is conjugated with DT or CRM 197, and no more than two saccharides are conjugated with TT. In a further embodiment, 19F is conjugated with DT or CRM197, and the rest of serotypes are divided between PD, TT and DT or CRM197. In a further embodiment, 19F is conjugated with DT or CRM197, and the rest of serotypes are divided between PD, TT and pneumolysin. In a further embodiment, 19F is conjugated with DT or CRM197, and the remaining serotypes are divided between PD, TT and CRM197. In a further embodiment, 19F is conjugated with dT or CRM197, and the remaining serotypes are divided between PD, TT, pneumolysin and optionally PhtD or the fusion protein PhtD / E. In a further embodiment 19F is conjugated with DT or CRM 197, 19A is conjugated with pneumolysin, an additional saccharide is conjugated with TT, an additional saccharide is conjugated with pneumolysin, 2 additional saccharides are conjugated with PhtD or PhtD / E and all saccharides additional ones are combined with PD.

In one embodiment, the immunogenic composition of the invention comprises protein D from Haemophilus influenzae. In this embodiment, if the PD is not one of the protein vehicles used to conjugate with any saccharide other than 19F, for example, 19F is conjugated with DT while the other serotypes are conjugated with one or more protein carriers that are not PD , then the PD will be present in the vaccine composition as a free protein. If the PD is one of the protein carriers used to conjugate saccharides other than 19F, then PD may optionally be present in the composition as a free protein.

The term "saccharide" throughout the present specification may indicate a polysaccharide or oligosaccharide and includes both. The polysaccharides are isolated from bacteria and can be modified in size to a certain extent by known methods (see, for example, EP497524 and EP497525) and optionally by microfluidization. The polysaccharides can be modified in size in order to reduce the viscosity in polysaccharide samples and / or to improve the filterability of conjugated products. Oligosaccharides have a small number of repeating units (typically 5-30 repeating units) and are usually hydrolyzed polysaccharides.

The capsular polysaccharides of Streptococcus pneumoniae comprise repeating oligosaccharide units which may contain up to 8 sugar moieties. For a review of the oligosaccharide units of the key serotypes of Streptococcus pneumoniae, see JONES, Christopher. Vaccines based on the cell surface carbohydrates of pathogenic bacteria. An. Acad. Bras. Ciénc., June 2005, vol. 77, n ° 2, p. 293-324. ISSN 0001-3765. In one embodiment, a capsular saccharide antigen may be a full length polysaccharide, however in others it may be an oligosaccharide unit, or shorter than a saccharide chain of native length of repeating units of oligosaccharides. In one embodiment all the saccharides present in the vaccine are polysaccharide. The full-length polysaccharides can be "resized", that is, their size can be reduced by various methods such as hydrolysis treatment, hydrogen peroxide treatment, size modification by emulsiflex® followed by peroxide treatment. hydrogen to generate oligosaccharide fragments or microfluidization.

The inventors have also pointed out that the focus of the technique has been to use oligosaccharides to facilitate the production of conjugates. The inventors have discovered that by using native or slightly modified polysaccharide conjugates, one or more of the following advantages can be achieved: 1) a conjugate having high immunogenicity that is filterable, 2) the ratio of polysaccharide to protein in the conjugate can be altered so that the ratio of polysaccharide to protein (w / w) in the conjugate can be increased (which can have an effect on the suppressive effect of the vehicle), 3) immunogenic conjugates that tend to hydrolysis they can be stabilized by the use of larger saccharides in conjugation. The use of larger polysaccharides may result in more crosslinking with the conjugate vehicle and may decrease the release of free saccharide from the conjugate. The conjugate vaccines described in the prior art tend to depolymerize the polysaccharides before conjugation in order to improve conjugation. The present inventors have found that saccharide conjugate vaccines that maintain a larger saccharide size can provide a good immune response against pneumococcal disease.

The immunogenic composition of the invention may therefore comprise one or more conjugates of saccharides in which the average size (weight average molecular weight; Pm) of each saccharide before conjugation is above 80 kDa, 100 kDa , 200 kDa, 300 kDa, 400 kDa, 500 kDa or 1000 kDa. In one embodiment the conjugate after conjugation should be easily filterable through a 0.2 micron filter so that a performance of more than 50, 60, 70, 80, 90 or 95 % post filtration compared to the pre-filtration sample.

For the purposes of the invention, "native polysaccharide" refers to a saccharide that has not been subjected to a process, the purpose of which is to reduce the size of the saccharide. A polysaccharide can become slightly reduced in size during normal purification procedures. Said saccharide is still native. Only if the polysaccharide has undergone size modification techniques would it be considered that the polysaccharide is not native.

For purposes of the invention "modified by a factor of up to x2" means that the saccharide is subjected to a process with the intention of reducing the size of the saccharide but maintaining a size greater than half the size of the native polysaccharide. x3, x4, etc., must be interpreted in the same way, that is, the saccharide is subjected to a procedure with the intention of reducing the size of the polysaccharide but maintaining a size greater than a third, a quarter, etc. of the size of the native polysaccharide. In one aspect of the invention, the immunogenic composition comprising Streptococcus pneumoniae saccharides of at least 10 serotypes conjugated to a protein carrier, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or each saccharide of S. pneumoniae is a native polysaccharide.

In one aspect of the invention, the immunogenic composition comprises Streptococcus pneumoniae saccharides of at least 10 serotypes conjugated to a protein carrier, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or each S. pneumoniae saccharide is modified in size by a factor of up to x2, x3, x4, x5, x6, x7, x8, x9 or x10. In one embodiment of this aspect most of the saccharides, for example 6, 7, 9 or more of the saccharides are modified in size by a factor of up to x2, x3, x4, x5, x6, x7, x8, x9 or x10 .

The molecular weight, or average molecular weight, of a saccharide of the present document refers to the average weight of the molecular weight (Pm) of the saccharide measured before conjugation as measured by MALLS.

The MALLS technique is well known in the art and is usually carried out as described in Example 2. For the MALLS analysis of pneumococcal saccharides, two columns (TSKG6000 and 5000PWxl) can be used in combination and the saccharides are eluted in water . The saccharides are detected using a light scattering detector (for example a Wyatt Dawn DSP equipped with a laser of 10 mW at 488 nm) and a thermometric refractometer (for example a Wyatt Otilab DSP equipped with a P100 cell and a red filter). 498 nm).

In one embodiment the saccharides of S. pneumoniae are native polysaccharides or native polysaccharides that have been reduced in size during a normal extraction procedure.

In one embodiment the S. pneumoniae saccharides are modified in size by mechanical cleavage, for example, by microfluidization or sonication. Microfluidization and sonication have the advantage of decreasing the size of the larger native polysaccharides sufficient to provide a filterable conjugate. The modification of the size is by a factor of no more than x20, x10, x8, x6, x5, x4, x3, or x2.

In one embodiment, the immunogenic composition comprises conjugates of S. pneumoniae that are produced from a mixture of native polysaccharides and saccharides that have been modified in size by a factor of not more than x20. In one aspect of this embodiment, most saccharides, for example 6, 7, 8 or more of the saccharides are modified in size by a factor of up to x2, x3, x4, x5, or x6.

In one embodiment, the saccharide of Streptococcus pneumoniae is conjugated to the protein carrier via a linker, for example a bifunctional linker. The linker is optionally heterobifunctional or homobifunctional, having for example a reactive amino group and a reactive carboxylic acid group, 2 reactive amino groups or two reactive carboxylic acid groups. The linker has, for example, between 4 and 20, 4 and 12, 5 and 10 carbon atoms. A possible link is ADH. Other linkers include B-propionamide (document W or 00/10599), nitrophenylethylamine (Gever et al., (1979) Med. Microbiol. Immunol. 165; 171-288), haloalkyl halides (US4057685), glycosidic linkages (documents US4673574 , US4808700), hexane diamine and 6-aminocaproic acid (US4459286). In one embodiment, ADH is used as a linker to conjugate the saccharide of serotype 18C.

The saccharide conjugates present in the immunogenic compositions of the invention can be prepared by any known coupling technique. The conjugation procedure could be based on the activation of the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated saccharide can therefore be coupled directly or via a spacer group (linker) to an amino group of the protein carrier. For example, the spacer could be cystamine or cysteamine to give a thiolated polysaccharide that could be coupled to the vehicle via a thioether link obtained after reaction with a maleimide-activated protein carrier (e.g. using GMBS) or a haloacetylated protein carrier (for example using iodoacetimide [for example, ethyl iodoacetimide HCL] or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP). Optionally, the cyanate ester (optionally produced by CDAP chemistry) is coupled with hexane diamine or ADH and the amino derivative saccharide is conjugated to the protein carrier using carbodiimide chemistry (eg, EDAC or EDC) by means of a carboxyl group of the protein vehicle Such conjugates are described in PCT published application WO 93/15760 Uniformed Services University and WO 95/08348 and WO 96/29094.

Other suitable techniques use carbodiimides, carbiinides, hydrazides, active esters, norborane, acid pnitrobenzoic, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in WO 98/42721. The conjugation may involve a carbonyl linker which can be formed by the reaction of a free hydroxyl group of the saccharide with CDI (Bethell et al., J. Biol. Chem. 1979, 254; 2572-4, Hearn et al., J. Chromatogr 1981. 218; 509-18) followed by reaction with a protein to form a carbamate linkage. This may involve reduction of the anomeric end in a primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form an intermediate CDI carbamate and coupling of the intermediate CDI carbamate with an amino group of a protein.

The conjugates can also be prepared by direct reductive amination procedures as described in US 4365170 (Jennings) and US 4673574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508.

A further process involves the coupling of an activated saccharide by cyanogen bromide (or CDAP) derived with adipic acid dihydrazide (ADH) with the protein carrier by carbodiimide condensation (Chu C. et al., Infect. Immunity, 1983245256), for example using Ed Ac.

In one embodiment, a hydroxyl group (optionally an activated hydroxyl group, for example, an activated hydroxyl group for making a cyanate ester [eg, using CDAP]) of a saccharide with an amino or carboxylic group of a direct protein is attached. or indirectly (by means of a linker). When a linker is present, the hydroxyl group of the saccharide is optionally linked with an amino group of a linker, for example using CDAP conjugation. An additional amino group in the linker (eg, ADH) that can be conjugated to a carboxylic acid group of a protein, for example using carbodiimide chemistry, for example using EDAC. In one embodiment, the pneumococcal capsular saccharides are conjugated to the linker first before the linker is conjugated to the protein carrier. Alternatively, the linker can be conjugated to the carrier prior to conjugation with the saccharide.

A combination of techniques can also be used, some saccharide-protein conjugates being prepared by CDAP, and some by reductive amination.

In general, the following types of chemical groups of a protein carrier can be used for coupling / conjugation:

A) Carboxyl (for example by means of aspartic acid or glutamic acid). In one embodiment this group is attached to the amino groups of the saccharides directly or to an amino group of a linker with carbodiimide chemistry, for example with EDAC.

B) Amino group (for example, by means of lysine). In one embodiment, this group is attached to carboxyl groups of the saccharides directly or to a carboxyl group in a linkage with carbodiimide chemistry, for example, with EDAC. In another embodiment this group binds to hydroxyl groups activated with CDAP or CNBr in the saccharides directly or to said groups in a linker; to saccharides or linkers that have an aldehyde group; to saccharides or linkers having an ester group of succinimide.

C) Sulfhydryl (for example, by means of cysteine). In one embodiment this group is attached to a saccharide or bromine linker or acetylated chlorine with maleimide chemistry. In one embodiment, this group is activated / modified by bis diazobenzidine.

D) Hydroxyl group (for example, by means of tyrosine). In one embodiment this group is activated / modified with bisdiazobenzidine.

E) Imidazolyl group (for example, by means of histidine). In one embodiment, this group is activated / modified with bis-diazobenzidine.

F) Guanidyl group (for example, by means of arginine).

G) Indolyl group (for example, by means of tryptophan).

In a saccharide, in general the following groups can be used for a coupling: OH, COOH or NH 2 . Aldehyde groups can be generated after different treatments known in the art such as: periodate, acid hydrolysis, hydrogen peroxide, etc.

Direct coupling strategies:

Saccharide-OH CNBr or CDAP --------> NH2-Prot cyanate ester -> conjugate

Saccharide-aldehyde NH2-Prot -> Schiff base NaCNBH3 -> conjugate

Saccharide-COOH NH2-Prot EDAC -> conjugate

Saccharide-NH2 COOH-Prot EDAC -> conjugate

Indirect coupling strategies by means of a spacer (linker):

Saccharide-OH CNBr or CDAP ^ cyanate ester NH2 -NH2 ^ Saccharide - NH2 COOH - Prot EDAC ^ conjugate

Saccharide-OH CNBr or CDAP ^ cyanate ester NH2-SH ^ Saccharide-SH SH-Prot (Native protein with a cysteine exposed or obtained after modification of amino groups of the protein, for example, by SPDP) ^ Saccharide-SS- Prot

Saccharide-OH CNBr or CDAP ^ cyanate ester NH2-SH ^ Saccharide-SH maleimide-Prot (modification of amino groups) ^ conjugate

Saccharide-OH CNBr or CDAP ^ cyanate ester NH2 - SH ^ Saccharide-SH haloacetylated-Prot ^ Conjugate

Saccharide-COOH EDAC NH2 - NH2 ^ Saccharide - NH2 EDAC COOH-Prot ^ conjugate

Saccharide-COOH EDAC + NH2-SH ^ Saccharide-SH SH-Prot (Native protein with a cysteine exposed or obtained after amino acid modification of the protein, for example, by SPDP) ^ Saccharide-S-S-Prot

Saccharide-COOH EDAC + NH2-SH ^ Saccharide-SH maleimide-Prot (modification of amino groups) ^ conjugate

Saccharide-COOH EDAC NH2 - SH ^ Saccharide-SH haloacetylated-Prot ^ Conjugate

Saccharide-Aldehyde NH2 - NH2 ^ Saccharide - NH2 EDAC COOH-Prot ^ conjugate

Note: instead of EDAC as above, any carbodiimide can be used.

In summary, the types of chemical group of the protein carrier that can be used in general for coupling with a saccharide are amino groups (for example on lysine residues), COOH groups (for example on aspartic acid and glutamic acid residues) and SH groups (if accessible) (for example on cysteine residues).

Optionally the ratio of protein carrier to saccharide of S. pneumoniae is between 1: 5 and 5: 1; 1: 2 and 2.5: 1, 1: 1 and 2: 1 (w / w). In one embodiment, most conjugates, for example 6, 7, 8, 9 or more of the conjugates have a ratio of protein carrier to saccharide that is greater than 1: 1, eg, 1.1: 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1 or 1.6: 1.

In one embodiment, at least one S. pneumoniae saccharide is conjugated to a protein carrier via a linker using CDAP and EDAC. For example, 18C can be conjugated to a protein by means of a linker (for example those having two hydrazino groups at their ends such as ADH) using CDAP and EDAC as described above. When a linker is used, CDAP can be used to conjugate the saccharide to a linker and then EDAC can be used to conjugate the linker to the protein, or alternatively, EDAC can first be used to conjugate the linker to the protein, after which can be used CDAP to conjugate the linker to the saccharide.

In general, the immunogenic composition of the invention may comprise a dose of each saccharide conjugate of between 0.1 and 20 μg, 1 and 10 μg or 1 and 3 μg of saccharide.

In one embodiment, the immunogenic composition of the invention contains each of the capsular saccharides of S. pneumoniae at a dose between 0.1-20 jg; 0.5-10 jg; 0.5-5 jg or 1-3 jg of saccharide. In one embodiment, the capsular saccharides may be present at different dosages, for example, some capsular saccharides may be present at a dose of exactly 1 jg or some capsular saccharides may be present at a dose of exactly 3 jg. In one embodiment, the saccharides of serotypes 3, 18C and 19F (or 4, 18C and 19F) are present at a higher dose than other saccharides. In one aspect of this embodiment, serotypes 3, 18C and 19F (or 4, 18C and 19F) are present at a dose of about or exactly 3 jg while other saccharides in the immunogenic composition are present at a dose of about or exactly 1 jg.

"Around" or "approximately" are defined as 10% more or less than the figure given for the purposes of the invention.

In one embodiment, at least one of the capsular saccharides of S. pneumoniae is directly conjugated to a protein carrier. Optionally, the at least one of the capsular saccharides of S. pneumoniae is directly conjugated by CDAP. In one embodiment, most capsular saccharides, for example 5, 6, 7, 8, 9 or more are directly linked to the protein carrier by CDAP (see WO 95/08348 and WO 96/29094).

The immunogenic composition may comprise Streptococcus pneumoniae proteins , referred to herein as Streptococcus pneumoniae proteins of the invention. Said proteins can be used as protein carriers, or they can be present as free proteins, or they can be present as protein carriers and as free proteins. The Streptococcus pneumoniae proteins of the invention are They expose on the surface, at least during part of the life cycle of the pneumococcus, or they are proteins that are secreted or released by the pneumococcus. Optionally, the proteins of the invention are selected from the following categories, such as proteins having the Type II signal sequence motif, LXXC (where X is any amino acid, eg, Sp101), proteins having an LPXTG motif (where X is any amino acid, for example Sp128, Sp130), and toxins (for example, Ply). Examples of these categories (or motifs) are the following proteins, or immunologically functional equivalents thereof.

In one embodiment, the immunogenic composition of the invention comprises at least 1 protein that is selected from the group consisting of the family of the polyhistidine triad (PhtX), family of proteins that bind to choline (CbpX), CbpX chewed , LytX family, truncated LytX, chimeric proteins (or fusions) of truncated CbpX-truncated LytX, pneumolysin (Ply), PspA, PsaA, Sp128, Sp101, Sp130, Sp125, and Sp133. In a further embodiment, the immunogenic composition comprises 2 or more proteins that are selected from the group consisting of the family of polyhistidine triads (PhtX), family of choline binding proteins (CbpX), truncated CbpX, family LytX, Truncated LytX, chimeric proteins (or fusions) of truncated CbpX-truncated LytX, pneumolysin (Ply), PspA, PsaA and Sp128. In a further embodiment, the immunogenic composition comprises 2 or more proteins that are selected from the group consisting of the family of polyhistidine triads (PhtX), family of choline binding proteins (CbpX), truncated CbpX, family LytX, Truncated LytX, chimeric proteins (or fusions) of truncated CbpX-truncated LytX, pneumolysin (Ply) and Sp128.

The Pht family (polyhistidine triads) comprises the PhtA, PhtB, PhtD, and PhtE proteins. The family is characterized by a lipidation sequence, two domains separated by a proline-rich region and several triads of histidine, possibly involved in metal or nucleoside binding or enzymatic activity, (3-5) supercoiled regions, a conserved N-terminus and a heterogeneous C-terminus. It is present in all strains of pneumococci tested. Homologous proteins have also been found in other streptococci and neiserias. In one embodiment of the invention, the Pht protein of the invention is PhtD. It is understood, however, that the terms Pht A, B, D, and E refer to proteins having the sequences disclosed in the subsequent references as well as to the variants of natural (and artificial) origin thereof which have a homology of sequence with at least 90% identity with the proteins referred to. Optionally it is at least 95% identity or at least 97% identity.

With respect to PhtX proteins, PhtA is disclosed in WO 98/18930, and is also referred to as Sp36. As noted above, it is a protein of the polyhistidine triad family that has the type II signal motif of LXXC. PhtD is disclosed in WO 00/37105, and is also referred to as Sp036D. As noted above, it is also a protein of the polyhistidine triad family and has the type II signal motif LXXC. PhtB is revealed in the document, and is also referred to as Sp036B. Another member of the PhtB family is the C3 degradation polypeptide, as disclosed in WO 00/17370. This protein is also from the family of polyhistidine triads and has the type II signal motif LXXC. For example, an immunologically functional equivalent is the Sp42 protein disclosed in WO 98/18930. A truncated PhtB (approximately 79 kD) is disclosed in WO99 / 15675 which is also considered a member of the PhtX family. PhtE is disclosed in WO00 / 30299 and referred to as BVH-3. When any Pht protein is referred to herein, it dignifies that the immunogenic fragments or fusions thereof of the Pht protein can be used. For example, a reference to PhtX includes the immunogenic fragments or fusions thereof of any Pht protein. A reference to PhtD or PhtB also refers to PhtDE or PhtBE fusions as found, for example, in WO0198334.

Pneumolysin is a multifunctional toxin with different cytolytic (hemolytic) and complement activation activities (Rubins et al., Am. Respi. Cit Care Med, 153: 1339-1346 (1996)). The toxin is not secreted by pneumococci, but is released when pneumococci are lysed by the influence of an autolysin. Its effects include, for example, the stimulation of the production of inflammatory cytokines by human monocytes, the inhibition of the pulsation of the cilia of the human respiratory epithelium, and the decrease of the bactericidal activity and the migration of neutrophils. The most obvious effect of pneumolysin is the lysis of red blood cells, which involves binding to cholesterol. Because it is a toxin, it is necessary to detoxify it (ie, it is not toxic to a human being when provided as an adequate dosage for protection) before it can be administered in vivo. The expression and cloning of wild type or native pneumolysin is known in the art. See for example, Walker et al., (Infect Immun, 55: 1184-1189 (1987)), Mitchell et al., (Biochim Biophys Acta, 1007: 67-72 (1989) and Mitchell et al., (NAR, 18: 4010 (1990).) Ply detoxification can be carried out by chemical means, for example, by subjecting it to formalin or glutaraldehyde treatment or a combination of both (documents W 04081515, PCT / EP2005 / 010258). Such methods are well known in the art for various toxins Alternatively, the Ply can be genetically detoxified.Therefore, the invention encompasses derivatives of pneumococcal proteins which may be, for example, mutated proteins. used herein means a molecule that has been subjected to elimination, addition or substitution of one or more amino acids using techniques well known by site-directed mutagenesis or any other conventional method, for example, as described above, it can be ede altering a mutant Ply protein so that it is biologically inactive while maintaining its immunogenic epitopes, see, for example, WO90 / 06951, Berry et al., (Infect Immun, 67: 981-985 (1999)) and the WO99 / 03884.

As used herein, it is understood that the term "Ply" refers to mutated or detoxified pneumolysin suitable for medical (ie, non-toxic) use.

As far as the family of the choline binding protein (CbpX) is concerned, members of that family were originally identified as pneumococcal proteins that could be purified by choline affinity chromatography. All choline binding proteins are non-covalently bound to phosphorylcholine residues of teichoic acid from the cell wall and lipoteichoic acid associated with the membrane. Structurally, they have several regions in common throughout the family, although the exact nature of the proteins (amino acid sequence, length, etc.) may vary. In general, choline-binding proteins comprise an N (N) terminal region, conserved repeat regions (R1 and / or R2), a proline-rich region (P) and a conserved choline-binding region (C) , which is composed of multiple repeats, comprising approximately half of the protein. As used in the present application, the expression "family of choline binding proteins (CbpX)" is selected from the group consisting of Choline binding proteins as identified in WO97 / 41151, PbcA, SpsA, PspC, CbpA, CbpD, and CbpG. CbpA is disclosed in WO97 / 41151. CbpD and CbpG are disclosed in document w O00 / 29434. PspC is disclosed in WO97 / 09994. PbcA is disclosed in WO98 / 21337. SpsA is a choline binding protein disclosed in WO 98/39450. Optionally the choline binding proteins are selected from the group consisting of CbpA, PbcA, SpsA and PspC.

An embodiment of the invention comprises truncated CbpXs in which "CbpX" is defined above and "truncated" refers to CbpX proteins lacking 50% or more of the choline binding region (C). Optionally, said proteins lack the complete choline binding region. Optionally, said truncated proteins lack (i) the choline binding region and (ii) a part of the N-terminal half of the protein as well, although it maintains at least one repeat region (R1 or R2). Optionally, the truncated has 2 repeated regions (R1 and R2). Examples of such embodiments are NR1xR2 and R1xR2 as illustrated in WO99 / 51266 or WO99 / 51188, however, other choline binding proteins lacking a similar choline binding region are also contemplated in the scope of the present invention.

The LytX family is formed by membrane-associated proteins that are associated with cell lysis. The N-terminal domain comprises a choline-binding domain (s), however the LytX family does not have all the characteristics found in the CbpA family noted above and therefore, for the present invention, the LytX family is considered other than the CbpX family. In contrast to the CbpX family, the C terminal domain contains the catalytic domain of the LytX protein family. The family comprises LytA, B and C. With respect to the LytX family, LytA is disclosed in Ronda et al., Eur J Biochem, 164: 621-624 (1987). LytB is disclosed in WO 98/18930, and which is also referred to as Sp46. LytC is also disclosed in WO 98/18930, and which is also referred to as Sp91. One embodiment of the invention comprises LytC.

Another embodiment comprises truncated LytXs in which "LytX" is defined above and "truncated" refers to LytX proteins that lack 50% or more of the choline binding region. Optionally said proteins lack the complete choline binding region. In yet another embodiment of the present invention, it comprises chimeric proteins (or fusions) of truncated truncated CbpX-LytX. Optionally it comprises NR1xR2 (or R1xR2) of CbpX and the terminal part C (Cterm, ie lacking the choline binding domains) of LytX (for example LytCCterm or Sp91 Cterm). Optionally, CbpX is selected from the group consisting of CbpA, PbcA, SpsA and PspC. Optionally, it is CbpA. Optionally, LytX is LytC (also referred to as Sp91). Another embodiment of the present invention is PspA, or truncated PsaA that lacks the choline binding domain (C) and that is expressed as a fusion protein with LytX. LytX is optionally LytC.

With respect to PsaA and PspA, both are known in the art. For example, PsaA and transmembrane deletion variants thereof have been described by Berry and Paton, Infect Immun 1996 Dec; 64 (12): 5255-62. PspA and transmembrane elimination variants thereof have been disclosed, for example, in US 5804193, WO 92/14488, and WO 99/53940.

Sp128 and Sp130 are disclosed in WO00 / 76540. Sp125 is an example of a pneumococcal surface protein with the cell wall anchored motif of LPXTG (where X is any amino acid). Any protein in this class of pneumococcal surface proteins with this motif has been found to be useful in the context of the present invention, and is therefore considered as an additional protein of the invention. Sp125 itself is disclosed in WO 98/18930, and is also known as ZmpB - a zinc metalloproteinase. The Sp101 is disclosed in WO 98/06734 (in which it has the reference number y85993). It is characterized by a Type I signal sequence. Sp133 is disclosed in WO 98/06734 (in which it has the reference number y85992). It is also characterized by a Type I signal sequence.

Examples of the Moraxella catarrhalis protein antigens that can be included in a vaccine combination (especially for the prevention of otitis media) are: OMP106 [WO 97/41731 (Antex) and WO 96/34960 (PMC)]; OMp21 or fragments thereof (WO 0018910); LbpA and / or LbpB [WO 98/55606 (PMC)]; TbpA and / or TbpB [WO 97/13785 and WO 97/32980 (PMC)]; CopB [Helminen ME, et al., (1993) Infect. Immun. 61: 2003-2010]; UspA1 and / or UspA2 [WO 93/03761 (University of Texas)]; OmpCD; HasR (PCT / EP99 / 03824); PilQ (PCT / EP99 / 03823); OMP85 (PCT / EP00 / 01468); lipo06 (GB 9917977.2); lipo10 (GB 9918208.1); lipo11 (GB document 9918302.2); lipo18 (GB 9918038.2); P6 (PCT / EP99 / 03038); D15 (PCT / EP99 / 03822); OmplAI (document PCT / EP99 / 06781); Hly3 (PCT / EP99 / 03257); and OmpE. Examples of non-typable Haemophilus influenzae antigens or fragments thereof can be included in a vaccine combination (especially for the prevention of otitis media) include: Fimbrin Protein [(US 5766608 - Ohio State Research Foundation)] and fusions comprising peptides the same [for example, peptide fusions LB1 (f); US 5843464 (OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [document Ep 281673 (State University of New York)]; TbpA and / or TbpB; Hia; Hsf; Hin47; Hif; Hmw1; Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94/12641); P2; and P5 (WO 94/26304).

The proteins of the invention can also be beneficially combined. By "combined" it is meant that the immunogenic composition comprises all the proteins of the following combinations. either as protein vehicles or as free proteins or a mixture of the two. For example, in a combination of two proteins as discussed hereafter, both proteins can be used as protein carriers, or both proteins can be present as free proteins, or both can be present as a carrier and as free protein, or a it may be present as a protein carrier and as a free protein while the other is present only as a protein carrier or only as a free protein, or one may be present as a protein carrier and the other as a free protein. When a combination of three proteins is given, similar possibilities exist. Combinations include, but are not limited to PhtD NR1xR2, PhtD NR1xR2-Sp91Cterm chimeric or fusion proteins, PhtD Ply, PhtD Sp128, PhtD PsaA, PhtD PspA, PhtA NR1xR2, PhtA NR1 xR2-Sp91 Chimeric Cterm or fusion proteins, PhtA Ply, PhtA Sp128, PhtA PsaA, PhtA PspA, NR1xR2 LytC, NR1xR2 PspA, NR1xR2 PsaA, NR1xR2 Sp128, R1xR2 LytC, R1xR2 PspA, R1xR2 PsaA, R1xR2 Sp128, R1xR2 PhtD, R1xR2 PhtA. Optionally, NR1xR2 (or R1xR2) is of CbpA or PspC. Optionally it is CbpA. Other combinations include combinations of 3 proteins such as PhtD NR1xR2 Ply, and PhtA NR1xR2 PhtD. In one embodiment, the vaccine composition comprises detoxified pneumolysin and PhtD or PhtDE as protein carriers. In a further embodiment, the vaccine composition comprises detoxified pneumolysin and PhtD or PhtDE as free proteins.

In an independent aspect, the present invention provides an immunogenic composition comprising at least four capsular saccharide conjugates of S. pneumoniae containing saccharides of different serotypes of S. pneumoniae in which at least one saccharide is conjugated with PhtD or a protein of fusion thereof and the immunogenic composition is capable of producing an effective immune response against PhtD.

An effective immune response against PhtD or fusion protein thereof is measured for example by a protection assay such as that described in example 15. An effective immune response provides at least 40%, 50%, 60% 70 %, 80% or 90% survival 7 days after the challenge with a heterologous strain. Because the challenge strain is heterologous, the protection that is achieved is due to the immune response against PhtD or the fusion protein thereof.

Alternatively, an effective immune response against PhtD is measured by ELISA as described in example 14. An effective immune response gives an anti-PhtD IgG response of at least 250, 300, 350, 400, 500, 550 or 600 | jg / ml GMC.

For example, the immunogenic composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 capsular saccharides of S. pneumoniae of different serotypes conjugated with PhtD or a fusion protein thereof. For example, additional serotypes 22F and 1, 2, 3, 4, 5, 6, or 7 selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 23F and 33F are conjugated with PhtD. In one embodiment two or three of serotypes 3, 6A and 22F are conjugated to PhtD or a fusion protein thereof.

In one embodiment, the immunogenic composition of the invention comprises at least one capsular saccharide of S. pneumoniae conjugated to PhtD or a fusion protein thereof by means of a linker, for example ADH. In one embodiment, one of the conjugation chemistries listed below is used.

In one embodiment, the immunogenic composition of the invention comprises at least one capsular saccharide of S. pneumoniae conjugated to a PhtD or a fusion protein thereof, wherein the ratio of PhtD to the saccharide in the conjugate is between 6: 1 and 1: 5, 6: 1 and 2: 1, 6: 1 and 2.5: 1, 6: 1 and 3: 1, 6: 1 and 3.5: 1 (w / w) or is greater than (ie, it contains a higher proportion of PhtD) 2.0: 1.2.5: 1, 3.0: 1, 3.5: 1 or 4.0: 1 (w / w).

In one embodiment, the immunogenic composition of the invention comprises pneumolysin.

The present invention further provides a vaccine containing the immunogenic compositions of the invention and a pharmaceutically acceptable excipient. The present invention further provides a method for making a vaccine comprising the step of mixing the immunogenic composition of the invention with a pharmaceutically acceptable excipient.

The vaccines of the present invention can be adjuvanted, particularly when it is intended to be used in an elderly population but also for use in infant populations. Suitable adjuvants include aluminum salts such as aluminum hydroxide gel or aluminum or alumina phosphate, but may also be other metal salts such as calcium, magnesium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationic or anionically derived saccharides, or polyphosphazenes.

The adjuvant is optionally selected to be a preferential inducer of a TH1 response type. Such high levels of Th1-type cytokines tend to favor the induction of cell-mediated immune responses for a given antigen, while high levels of Th2-type cytokines tend to favor the induction of humoral immune responses to the antigen.

The distinction between Th1 and Th2 immune responses is not absolute. In reality an individual will have an immune response that is described as being predominantly Th1 or predominantly Th2. However, it is often convenient to consider cytokine families in terms of what they describe in CD4 + T lymphocyte clones by Mosmann and Coffman (Mosmann, TR and Coffman, RL (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead To different functional properties (Annual Review of Immunology, 7, p145-173) Traditionally, Th1-type responses are associated with the production of INF-y and cytokines IL-2 by T lymphocytes. Other cytokines are often associated directly. with the induction of Th1 type immune responses are not produced by T lymphocytes, such as IL-12, On the contrary, Th2 type responses are associated with the secretion of IL-4, IL-5, IL-6, IL- 10. Suitable adjuvant systems that promote a predominantly Th1 response include: Monophosphoryl lipid A or a derivative thereof (or lipid A detoxified in general - see for example WO2005107798), particularly monophosphoryl lipid A 3-de-O-acyl (3D-MPL) (for preparation see GB 2220211 A); and a combination of monophosphoryl lipid A, optionally A3-des-O-acylated monophosphoryl lipid together with an aluminum salt (for example aluminum phosphate or aluminum hydroxide) or an oil in water emulsion. In such combinations, the antigen and 3D-MPL are contained in the same particulate structures, allowing a more efficient delivery of the antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is capable of further enhancing the immunogenicity of an antigen adsorbed to alumina [Thoelen et al., Vaccine (1998) 16: 708-14; EP 689454-B1].

An improved system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL is disclosed in WO 94/00153, or a less reactogenic composition where QS21 is inactivated with cholesterol as discloses in WO 96/33739. A particularly potent adjuvant formulation involves QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210. In one embodiment the immunogenic composition additionally comprises a saponin, which may be QS21. The formulation may also comprise an oil in water emulsion and tocopherol (WO 95/17210). Oligonucleotides containing non-methylated CpG (WO 96/02555) and other immunomodulatory oligonucleotides (WO0226757 and WO03507822) are also preferential inducers of a Th1 response and are suitable for use in the present invention.

Particular adjuvants are those selected from the group of metal salts, oil-in-water emulsions, Toll-like receptor agonists (in particular the Toll-like receptor 2 agonist, Toll-like receptor 3 agonist, receptor 4 agonist). Toll type, agonist of Toll-like receptor 7, receptor agonist 8 type Toll and receptor agonist 9 type Toll), saponins and combinations thereof.

An adjuvant that can be used with the vaccine compositions of the invention are preparations in ampoules or vesicles of the outer membrane of Gram-negative bacterial strains such as those taught in WO02 / 09746 - particularly, ampoules of N. meningitidis. The adjuvant properties of the ampoules can be improved by retention of LOS (lipooligosaccharides) on their surface (for example, by extraction with low concentrations of detergent [eg 0-0.1% deoxycholate]). LOS can be detoxified by means of mutations msbB (-) or htrB (-) which are treated in WO02 / 09746. The adjuvant properties can be improved by retaining PorB (and optionally eliminating PorA) from the meningococcal bullae. The adjuvant properties can also be improved by truncating the outer structure of the LOS saccharide core in the meningococcal bullae - for example by means of an IgtB (-) mutation as discussed in WO2004 / 014417. Alternatively, the aforementioned LOSs (eg isolates of a msbB (-) strain and / or IgtB (-)) can be purified and used as an adjuvant in the compositions of the invention.

An additional adjuvant that can be used with the compositions of the invention can be selected from the group: a saponin, lipid A or a derivative thereof, an immunostimulatory oligonucleotide, an alkyl glucosaminide phosphate, an oil in water emulsion or combinations of them. An additional adjuvant that can be used with the compositions of the invention is a metal salt in combination with another adjuvant. In one embodiment, the adjuvant is a Toll-like receptor agonist in particular an agonist of a 2, 3, 4, 7, 8 or 9 Toll-like receptor or a saponin, in particular QS21. In one embodiment, the adjuvant system comprises two or more adjuvants of the above list. In particular, the combinations optionally contain a saponin (in particular QS21) adjuvant and / or a Toll-like receptor agonist 9 such as an immunostimulatory oligonucleotide containing CpG. Other combinations comprise a saponin (in particular QS21) and a Toll-like receptor 4 agonist such as monophosphoryl lipid A or its deacylated derivative, 3D-MPL, or a saponin (in particular QS21) and a Toll-like receptor 4 ligand such as an alkyl glucosaminide phosphate.

In one embodiment, the adjuvants are combinations of 3D-MPL and QS21 (EP 0 671 948 B1), oil-in-water emulsions comprising 3D-MPL and QS21 (documents w O 95/17210, WO 98/56414), or 3D-MPL formulated with other vehicles (EP 0 689 454 B1). In one embodiment, the adjuvant systems they comprise a combination of 3D-MPL, QS21 and a CpG oligonucleotide as described in US6558670, US6544518.

In one embodiment, the adjuvant is a Toll-like receptor 4 (TLR) ligand, optionally an agonist such as a lipid A derivative, particularly monophosphoryl lipid A or more particularly 3-deacylated monophosphoryl lipid A (3D-MPL).

3D-MPL is available from Glaxo SmithKline Biologicals North America and primarily promotes CD4 + T cell responses with an IFN-g (Th1) phenotype. This can be produced according to the methods disclosed in GB 2220211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5, or 6 acylated chains. in one embodiment, the compositions of the present invention utilize small particle 3D-MPL. The small particle of 3D-MPL has a particle size so that it can be sterilized by filtering through a 0.22 mm filter. Said preparations are described in International Patent Application No. WO 94/21292. Synthetic lipid A derivatives are known and thought to be TLR 4 agonists, but are not limited to:

OM174 (2-deoxy-6-o- [2-deoxy-2 - [(R) -3-dodecanoyloxytetra-decanoylamino] -4-o-phosphono-pD-glucopyranosyl] -2 - [(R) -3-hydroxytetradecanoylamino ] -a-glucopyranosyl dihydrogen phosphate), (WO 95/14026)

OM 294 DP (3S, 9R) -3 - [(R) -dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 (R) - [(R) -3-hydroxytetradecanoylamino] decan-1,10-diol, 1 , 10-bis (dihydrogen phosphate) (WO99 / 64301 and W 00 / o462)

OM 197 MP-Ac DP (3S-, 9R) -3 - [(R) -dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 - [(R) -3-hydroxytetradecanoyl amino] decan-1,10-diol , 1-dihydrogen phosphate 10- (6-aminohexanoate) (WO 01/46127)

Other TLR 4 ligands that can be used are alkyl glucosaminide phosphates (AGP) such as those disclosed in WO9850399 or US6303347 (also disclosed are methods for the preparation of AGP), or pharmaceutically acceptable AGP salts as disclosed. in document US6764840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists. It is thought that both are useful as adjuvants.

Another immunostimulant for use in the present invention is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quilaja Saponaria Molina and was described for the first time as having adjuvant activity by Dalsgaard et al., In 1974 ("Saponin adjuvants", Archiv. Fur die gesamte Virusforschung, Vol.

44, Springer Verlag, Berlin, p243-254). The purified fragments of Quil A have been isolated by HPLC which maintains the adjuvant activity without the toxicity associated with Quil A (EP 0 362 278), for example, QS7 and QS21 (also known as QA7 and QA21). QS21 is a saponin derived from the bark of Quillaja saponaria Molina that induces CD8 + cytotoxic T lymphocytes (CTL), Th1 cells and a predominant antibody response in IgG2a and is a saponin in the context of the present invention.

Particular formulations of QS21 have been described which are an embodiment of the invention, these formulations additionally comprise a sterol (WO96 / 33739). The saponins which form part of the present invention can be separated in the form of micelles, mixed micelles (optionally with bile salts) or can be in the form of iSCOM matrices (EP 0 109 942 B1), liposomes or related colloidal structures such as complexes multimeric worm-type or ring-type or lipid / laminate structures and lamellae when formulated with cholesterol and lipids, or in the form of an oil-in-water emulsion (for example as in WO 95/17210). Saponins can be associated with a metal salt, such as aluminum hydroxide, or aluminum phosphate (W document or 98/15287). Optionally, the saponin is presented in the form of a liposome, ISCOM or in an oil-in-water emulsion.

An improved system involves the combination of a monophosphoryl lipid A (or detoxified lipid A) and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition in the that QS21 is inactivated with cholesterol as disclosed in WO 96/33739. A particularly potent adjuvant formulation involves tocopherol with or without QS21 and / or 3D-MPL in an oil-in-water emulsion which is described in WO 95/17210. In one embodiment the immunogenic composition additionally comprises a saponin, which may be QS21.

Immunostimulatory oligonucleotides or any other Toll-like receptor (TLR) can also be used. Oligonucleotides for use in the adjuvants or vaccines of the present invention are optionally oligonucleotides containing CpG, optionally containing two or more CpG dinucleotide motifs separated by at least three, optionally at least six or more nucleotides. A CpG motif is a nucleotide Cytosine followed by a Guanine nucleotide. The CpG oligonucleotides of the present invention are typically deoxynucleotides. In one embodiment, the internucleotide in the oligonucleotide is phosphorodithioate, or a phosphorothioate linkage, although phosphodiester linkages and other internucleotide linkages are within the scope of the invention. Oligonucleotides with mixed internucleotide linkages are also included in the scope of the invention. Methods for producing phosphorothioate or phosphorodithioate oligonucleotides are described in US 5,666,153, US 5,278,302 and WO95 / 26204.

Examples of oligonucleotides have the following sequences. The sequences optionally contain modified phosphorothioate internucleotide linkages.

OLIGO 1 (SEQ ID NO: 1): TCC ATG ACG TTC CTG ACG TT (CpG 1826)

OLIGO 2 (SEQ ID NO: 2): TCT CCC AGC GTG CGC CAT (CpG 1758)

OLIGO 3 (SEQ ID NO: 3): ACC GAT GAC GTC GCC GGT GAC GGC ACC

ACG OLIGO 4 (SEQ ID NO: 4): TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006)

OLIGO 5 (SEQ ID NO: 5): TCC ATG ACG TTC CTG ATG CT (CpG 1668)

OLIGO 6 (SEQ ID NO: 6): TCG ACG TTT TCG GCG CGC GCC G (CpG 5456)

Alternative CpG oligonucleotides can comprise the above sequences in which they have deletions or additions without consequences therein.

The CpG oligonucleotides that are used in the present invention can be synthesized by any method known in the art (for example, see EP 468520). Conveniently, said oligonucleotides can be synthesized using an automated synthesizer.

The adjuvant may be an oil-in-water emulsion or may comprise an oil-in-water emulsion in combination with other adjuvants. The oil phase of the emulsion system optionally comprises a metabolisable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as "that is capable of being transformed by metabolism" (Dorland's Illustrated Medical Dictionary, W.B. Sanders Company, 25th edition (1974)). The oil can be any vegetable oil, fish oil, animal or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, and grains are common sources of vegetable oils. Synthetic oils are also part of the invention and may include commercially available oils such as NEOBEE® and others. Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetraicosahexaene) is an unsaturated oil found in large quantities in shark liver oil, and in small amounts in olive oil, wheat germ oil, rice bran oil, and yeast, and is an oil for use in the present invention. Squalene is a metabolizable oil thanks to the fact that it is an intermediate in cholesterol biosynthesis (Merck index, 10th Edition, entry n ° 8619).

Tocols (for example, vitamin E) are often also used in oil emulsion adjuvants (EP 0382 271 B1; US5667784; WO 95/17210). The tocols which are used in the oil emulsions (optionally in oil-in-water emulsions) of the invention can be formulated as described in EP 0382 271 B1, in which the tocols can be dispersions of tocol droplets, optionally they comprise an emulsifier, optionally less than 1 micron. Alternatively, the tocols can be used in combination with another oil to form the oil phase of an oil emulsion. Examples of oily emulsions that can be used in combination with tocol are described herein, such as the metabolizable oils described above.

It has been suggested that oil-in-water adjuvants per se are useful as adjuvant compositions (EP 0 399 843B), combinations of oil-in-water emulsions and other active agents for vaccines have also been described (WO 95/17210; WO 98/56414, WO 99/12565, WO 99/11241). Other oil emulsion adjuvants have been described, such as water-in-oil emulsions (US 5,422,109, EP 0480982 B2) and water-in-oil-in-water emulsions (US 5,424,067, EP 0480981 B). All of which form oil emulsion systems (particularly when incorporating tocols) to form adjuvants and compositions of the present invention.

In one embodiment, the oil emulsion (e.g., oil-in-water emulsions) further comprises an emulsifier such as TWEEN80 and / or a sterol such as cholesterol.

In one embodiment, the oil emulsion (optionally an oil in water emulsion) comprises a non-toxic metabolizable oil, such as squalene, squalene or a tocopherol such as alpha tocopherol (and optionally both squalene and alpha tocopherol) and optionally a emulsifier (or surfactant) such as Tween 80. A sterol (eg, cholesterol) may also be included. The process for producing oil-in-water emulsions is well known to the person skilled in the art. Commonly, the method comprises mixing the oily phase containing tocol with a surfactant such as a PBS / TWEEN80 ™ solution, followed by homogenization using a homogenizer, it would be clear to one skilled in the art that a process comprising the mixing step twice through a syringe needle would be suitable to homogenize small volumes of liquid. Similarly, the emulsion procedure in a microfluidizer (M110S Microfluidic machine, maximum of 50 passages, for a period of 2 minutes at maximum inlet pressure of 6 bar (outlet pressure of approximately 850 bar)) could be adapted by the expert in the art to produce higher or lower emulsion volumes. The adaptation could be achieved by routine experimentation comprising the measurement of the resulting emulsion until a preparation with drops of oils of the necessary diameter is achieved. In an oil-in-water emulsion, the oil and the emulsifier should be in an aqueous vehicle. The aqueous vehicle can be, for example, phosphate buffered saline solution.

The size of the oil droplets found in the stable oil-in-water emulsion is optionally less than 1 micron, may be in the range of substantially 30-600 nm, optionally substantially about 30-500 nm in diameter, and optionally substantially 150-500 nm in diameter, and in particular about 150 nm in diameter as measured by photon correlation spectroscopy. In this regard, 80% of the oil drops per number should be in the ranges, optionally more than 90% and optionally more than 95% of the drops per number are in the defined size ranges. The amounts of components present in the oil emulsions of the present invention are conventionally in the range of from 0.5-20% or 2 to 10% oil (of the total volume of the dose), such as squalene; and when they are present from 2 to 10%% tocopherol alfa; and from 0.3 to 3% surfactant, such as polyoxyethylene sorbitan monooleate. Optionally, the ratio of oil (e.g., squalene): tocol (e.g. α-tocopherol) is equal to or less than 1, since this provides a more stable emulsion. An emulsifier, such as Tween 80 or Span 85 may also be present at a level of about 1%. In some cases it may be advantageous that the vaccines of the present invention may additionally contain a stabilizer.

Examples of emulsion systems are described in WO 95/17210, WO 99/11241 and WO 99/12565 which disclose emulsion adjuvants based on squalene, α-tocopherol, and TWEEN80, optionally formulated with the QS21 and / or 3D immunostimulants. -MPL. Therefore, in one embodiment of the present invention, the adjuvant of the invention may additionally comprise further immunostimulants, such as LPS or derivatives thereof and / or saponins. Examples of additional immunostimulants are described herein and / or in "Vaccine Design - The Subunit and Adjuvant Approach" 1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, MF, and Newman, MJ, Plenum Press, New York and London , ISBN 0-306-44867-X.

In one embodiment, the adjuvant and the immunogenic compositions according to the invention comprise a saponin (e.g., QS21) and / or an LPS derivative (e.g., 3D-MPL) in an oily emulsion as described above, optionally with a sterol (for example, cholesterol). Additionally, the oil emulsion (optionally an oil in water emulsion) may contain Span 85 and / or lecithin and / or tricaprylin. Adjuvants comprising an oil in water emulsion, a sterol and a saponin are described in WO 99/12565.

Typically, for administration to humans, saponin (for example QS21) and / or the LPS derivative (eg, 3D-MPL) will be present in a human dose of the present immunogenic composition in the range of 1 jg- 200 | jg, such as 10-100 | jg, or 10 jg-50 | jg per dose. Normally, the oil emulsion (optionally an oil in water emulsion) will comprise from 2 to 10% of metabolizable acetite. Optionally it will comprise from 1 to 10% of squalene, from 2 to 10% of alpha tocopherol and from 0.3 to 3% (optionally of 0.4-2%) of emulsifier (optionally Tween 80 [polyoxyethylated sorbitan monooleate ]). When squalene and alpha tocopherol are present, the squalene: alpha tocopherol ratio is optionally equal to or less than 1, since it provides a more stable emulsion. Span 85 (sorbitan trioleate) may also be present at a level of 0.5 to 1% in the emulsions used in the invention. In some cases it may be advantageous that the immunogenic compositions and vaccines of the present invention will additionally contain a stabilizer, for example other emulsifiers / surfactants, including caprylic acid (Merck Index 10th Edition, entry no. 1739), for example Tricaprylin.

When squalene and a saponin (optionally QS21) are included, it is beneficial to also include a sterol (optionally cholesterol) in the formulation since this allows a reduction in the total level of oil in the emulsion. This results in a reduction of the manufacturing cost, improves the convenience of vaccination, and also improves quantitatively and qualitatively the result of the immune responses, such as a better production of IFN-y. Accordingly, the adjuvant system of the present invention typically comprises a ratio of metabolizable oil: saponin (w / w) in the range of 200: 1 to 300: 1, the present invention can also be used in a "low in" form. oil "whose optional range is from 1: 1 to 200: 1, optionally from 20: 1 to 100: 1, or substantially 48: 1, this vaccine maintains the beneficial adjuvant properties of all components, with a level of reactogenicity very reduced. Accordingly, some embodiments have a squalene ratio: QS21 (w / w) in the range of 1: 1 to 250: 1, or 20: 1 to 200: 1, or 20: 1 to 100: 1, or substantially 48 :one. Optionally a sterol (eg, cholesterol) can also be included by being present in a ratio of saponin: sterol as described herein.

The emulsion systems of the present invention optionally have a small oil droplet size in the sub-micron range. Optionally, the oil drop sizes will be in the range of 120 to 750 nm, or from 120-600 nm in diameter.

A particularly potent formulation of adjuvant (for the latter combination with AIPO4 in the immunogenic compositions of the invention) involves a saponin (e.g., QS21), an LPS derivative (e.g., 3D-MPL) and an oily emulsion (e.g. , squalene and alpha tocopherol in an oil-in-water emulsion) as described in WO 95/17210 or in WO 99/12565 (in particular the adjuvant formulation 11 of Example 2, Table 1).

Examples of a TLR2 agonist include a peptidoglycan or lipoprotein. Imidazoquinolines, such as Imiquimod and Resiquimod are known LR7 t agonists. Also known is a TLR agonist which is a single-stranded RNA (TLR8 in humans and TLR7 in mice), whereas a double-stranded RNA and poly IC (polyinosin-polycytidyl acid - a synthetic mimic of a viral RNA) ), are examples of agonists TLR3. The 3D-MPL is an example of a TLR4 agonist while CPG is an example of a TLR9 agonist.

The immunogenic composition may comprise an antigen and an immunostimulant adsorbed to a metal salt. Aluminum-based vaccine formulations in which the 3-de-O-acylated monophosphoryl lipid A antigen and immunostimulant (3D-MPL) are adsorbed on the same particle are described in EP 0576478 B1, EP 0689454 B1, and EP 0633784 B1. In these cases then the antigen is first adsorbed to the aluminum salt followed by the adsorption of the 3D-MPL stimulant on the same aluminum particles. Said process first involves suspending 3D-MPL by sonication in a water bath until the particles reach a size between 80 and 500 nm. The antigen is normally adsorbed in the aluminum salt by stirring at room temperature for one hour. The 3D-MPL suspension is then added to the adsorbed antigen and the formulation is incubated at room temperature for one hour, and then maintained at 4 ° C until use.

In another method, the immunostimulant and the antigen are in separate metal particles, as described in EP 1126876. The improved process comprises the adsorption of the immunostimulant, on a metal salt particle, followed by the adsorption of the antigen on another particle of metallic salt, followed by mixing the separated metal particles to form a vaccine. The adjuvant for use in the present invention may be an adjuvant composition comprising an immunostimulant, adsorbed to a metal salt particle, characterized in that the metal salt particle is substantially free of another antigen. In addition, vaccines are provided by the present invention and are characterized in that the immunostimulant is adsorbed on metal salt particles that are substantially free of another antigen, and because the metal salt particles having the adsorbed antigen are substantially free of another immunostimulant. In consequence, the present invention provides an adjuvant formulation comprising an immunostimulant that has been adsorbed to a particle of a metal salt, which is characterized in that the composition is substantially free of another antigen. In addition, this adjuvant formulation can be intermediate which, if said adjuvant is used, is necessary for the manufacture of a vaccine. Accordingly, a method for the manufacture of a vaccine comprising the mixture of an adjuvant composition which is one or more immunostimulants adsorbed on a metal particle with an antigen is provided. Optionally, the antigen has been pre-adsorbed to a metal salt. Said metal salt may be identical or similar to the metal salt in which the immunostimulant has been adsorbed. Optionally, the metal salt is an aluminum salt, for example, aluminum phosphate or aluminum hydroxide.

The present invention additionally provides a vaccine composition comprising an immunostimulant adsorbed on a first particle of a metal salt, and an antigen adsorbed on a metal salt, characterized in that the first and second particles are separate particles.

The derivatives or mutations of LPS or LOS or lipid A derivatives that are described herein are designed to be less toxic (e.g., 3D-MPL) than the native lipopolysaccharides and are interchangeable equivalents with respect to any of the uses of These remains are described in this document.

In one embodiment the adjuvant that is used in the compositions of the invention comprises a liposomal carrier (produced by known techniques from phospholipids (such as dioleoylphosphatidyl choline [DOPC]) and optionally a sterol [such as cholesterol]). Such liposomal vehicles can harbor lipid A derivatives [such as 3D-MPL - see above] and / or saponins (such as QS2l - see above). In one embodiment the adjuvant comprises (per 0.5 ml dose) 0.1-10 mg, 0.2-7, 0.3-5, 0.4-2, or 0.5-1 mg (e.g. , 0.4-0.6, 0.9-1.1, 0.5 or 1 mg) of phospholipid (eg, DOPC), 0.025-2.5, 0.05-1.5, 0.075-0 , 75, 0.1-0.3, or 0.125 0.25 mg (eg, 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg) of sterols (e.g. cholesterol), 5-60, 10-50, or 20-30 | jg (for example, 5-15, 40-50, 10, 20, 30, 40 or 50 | jg) of a lipid A derivative (e.g. 3D-MPL), and 5-60, 10 50, or 20-30 jg (for example, 5-15, 40-50, 10, 20, 30, 40 or 50 jg) of saponin (e.g., QS21).

This adjuvant is particularly suitable for vaccine formulations for the elderly. In one embodiment the vaccine composition comprising this adjuvant comprises conjugates that are derived from at least all of the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more serotypes 3, 6A, 19A, and 22F), in which the GMC antibody titer induced against one or more (or all) of the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly lower than induced by the vaccine Prevnar® in vaccinated humans.

In one embodiment the adjuvant used for the compositions of the invention comprises an oil-in-water emulsion produced from a metabolizable oil (such as squalene), an emulsifier (such as Tween 80) and optionally a tocol (such as alpha tocopherol). . In one embodiment the adjuvant comprises (per 0.5 ml dose) 0.5-15, 1-13, 2-11, 4-8, or 5-6 mg (eg, 2-3, 5-6, or 10-11 mg) of metabolizable oil (such as squalene), 0.1-10, 0.3-8, 0.6-6, 0.9-5, 1-4, or 2-3 mg (per example, 0.9-1.1, 2-3 or 4-5 mg) of emulsifier (such as Tween 80) and optionally 0.5-20, 1-15, 2-12, 4-10, 5-7 mg (for example, 11-13, 5-6, or 2-3 mg) of tocol (such as alpha tocopherol).

This adjuvant may optionally further comprise 5-60, 10-50, or 20-30 jg (eg, 5-15, 40-50, 10, 20, 30, 40 or 50 jg) of lipid A derivative (e.g. , 3D-MPL).

These adjuvants are particularly suitable for formulations for children and the elderly. In an embodiment the The vaccine composition comprising this adjuvant comprises saccharide conjugates derived from at least all of the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more of serotypes 3, 6A, 19A, and 22F), in which the GMC antibody titer induced against one or more (or all) of the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly lower than that induced by the vaccine Prevnar® in vaccinated humans.

This adjuvant may optionally contain 0.025-2.5, 0.05-1.5, 0.075-0.75, 0.1-0.3, or 0.125-0.25 mg (eg, 0.2-0, 3, 0.1-0.15, 0.25 or 0.125 mg) of a sterol (eg, cholesterol), 5-60, 10-50, or 20-30 μg (eg, 5-15, 40 -50, 10, 20, 30, 40 or 50 jg) of a lipid A derivative (e.g., 3D-MPL), and 5-60, 10-50, or 20 30 jg (e.g., 5-15, 40-50, 10, 20, 30, 40 or 50 jg) of a saponin (e.g., QS21).

This adjuvant is particularly suitable for vaccine formulations for the elderly. In one embodiment the vaccine composition comprising this adjuvant comprises saccharide conjugates derived from at least all of the following serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more of serotypes 3, 6A, 19A, and 22F), in which the GMC antibody titer induced against one or more (or all) of the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly lower than induced by the Prevnar® vaccine in vaccinated humans.

In one embodiment the adjuvant that is used for the compositions of the invention comprises aluminum phosphate and a lipid A derivative (such as 3D-MPL). This adjuvant may comprise (per dose of 0.5 ml) 100-750, 200 500, or 300-400 g of Al as aluminum phosphate, and 5-60, 10-50, or 20-30 jg (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 jg) of a lipid A derivative (e.g., 3D-MPL).

This adjuvant is particularly suitable for vaccine formulations for the elderly or children. In one embodiment the vaccine composition comprising this adjuvant comprises saccharide conjugates derived from at least all of the following serotypes: 4, 6b, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also comprise one or more of serotypes 3, 6A, 19A, and 22F), in which the GMC antibody titer induced against one or more (or all) of the vaccine components 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly lower than induced by the Prevnar® vaccine in vaccinated humans.

Vaccine preparations containing immunogenic compositions of the present invention can be used to protect or treat a mammal susceptible to infection, by administering said vaccine by means of a systemic or mucosal route. These administrations may include injection by intramuscular, intraperitoneal, intradermal, or subcutaneous routes; or by means of mucosal administration in the oral and alimentary, respiratory, genitourinary tracts. Intranasal administration of vaccines for the treatment of pneumonia or otitis media is possible (since nasopharyngeal transport of pneumococci can be prevented more effectively, attenuating the infection in this way at its earliest stage). Although the vaccine of the invention can be administered as a single dose, the components thereof can be co-administered together at the same time or at different times (for example pneumococcal saccharide conjugates could be administered separately, at the same time or 1-2 weeks after the administration of any bacterial protein component of the vaccine for optimal coordination of immune responses with respect to one another). For co-administration, the optional Th1 adjuvant can be present in each or all of the different administrations. In addition to a single administration route, 2 different routes of administration can be used. For example, saccharides or saccharide conjugates can be administered IM (or ID) and bacterial proteins can be administered IN (or ID). In addition, the vaccines of the invention can be administered IM for the sensitization doses and IN for the booster doses.

The content of antigenic proteins in the vaccine will normally be in the range of 1-100 jg, optionally 5-50 jg, more typically in the range of 5-25 jg. After an initial vaccination, subjects may receive one or more booster immunizations spaced appropriately.

The preparation of vaccines is generally described in Vaccine Design ("The subunit and adjuvant approach" (eds Powell M.F. and Newman M.J.) (1995) Plenum Press New York). Encapsulation in liposomes is described by Fullerton, U.S. Pat. UU 4,235,877.

The vaccines or immunogenic compositions of the present invention can be stored in solution or lyophilized. In one embodiment, the solution is lyophilized in the presence of sugar which acts as an amorphous lyoprotectant, such as sucrose, trehalose, glucose, mannose, maltose or lactose. In one embodiment, the solution is lyophilized in the presence of a sugar that acts as an amorphous lyoprotectant, and a bulking agent that provides a better agglutinated structure such as glycine or mannitol. The presence of a crystalline volume agent allows the shortening of the freeze drying cycles, in the presence of a high salt concentration. Examples of such mixtures for use in the lyophilization of immunogenic compositions or vaccines of the invention include sucrose / glycine, trehalose / glycine, glucose / glycine, mannose / glycine, maltose / glycine, sucrose / mannitol, trehalose / mannitol, glucose / mannitol, mannose / mannitol and maltose / mannitol. Normally, the molar ratio of the two constituents is optionally 1: 1, 1: 2, 1: 3, 1: 4, 1: 5 or 1: 6. The immunogenic compositions of the invention optionally comprise the lyophilization reagents described above.

The above stabilizing agents and mixtures of stabilizing agents may additionally include a polymer capable of increasing the crystallization transition temperature (Tg) of the formulation, such as poly (vinylpyrrolidone) (PVP), hydroxyethyl starch or dextran, or a polymer which it acts as a crystalline volume agent such as polyethylene glycol (PEG) for example having a molecular weight between 1500 and 6000 and dextran.

The immunogenic compositions of the invention are optionally lyophilized and reconstituted extemporaneously before use. Lyophilization can result in a more stable composition (vaccine) and possibly lead to higher antibody titers in the presence of 3D-MPL and in the absence of an aluminum-based adjuvant.

In one aspect of the invention, a vaccine kit is provided, comprising a vial containing an immunogenic composition of the invention, optionally in lyophilized form, and further comprising a vial containing an adjuvant as described herein. It is anticipated that in this aspect of the invention, the adjuvant will be used to reconstitute the lyophilized immunogenic composition.

Although the vaccines of the present invention can be administered by any route, the administration of the described skin (ID) vaccines forms an embodiment of the present invention. Human skin includes a "keratinized" outer cuticle, called the stratum corneum, that covers the epidermis. Beneath this epidermis is a layer called the dermis, which in turn covers the subcutaneous tissue. Researchers have shown that the injection of a vaccine into the skin, and in particular the dermis, stimulates the immune response, which can also be associated with several additional benefits. Intradermal vaccination with the vaccines described herein is an optional feature of the present invention.

The conventional technique of intradermal injection, the "Mantoux procedure", comprises the steps of cleaning the pial, and then pinching the skin with one hand, and with the bevel of a narrow gauge needle (caliber 26-31) locating the needle upwards insert it at an angle of between 10-15 °°. Once the bevel of the needle is inserted, the cone of the needle is lowered and further advanced while providing a small pressure to raise it under the skin. The liquid is then injected very slowly thereby forming a blister or bump on the surface of the skin, followed by a slow removal of the needle.

More recently, devices designed specifically to administer liquid agents in or through the skin have been described, for example the devices described in WO 99/34850 and EP 1092444, also the jet injection devices described for example in WO documents. 01/13977; US 5,480,381, US 5,599,302, US 5,334,144, US 5,993,412, US 5,649,912, US 5,569,189, US 5,704,911, US 5,383,851, US 5,893,397, US 5,366,120, US 5,339,163, US 5,312,335, US 5,503,627, US 5,064,413, US 5,520, 639, US 4,596,556, US 4,790,824, US 4,941,880, US 4,940,460, WO 97/37705 and WO 97/13537. Alternative methods of intradermal administration of vaccine preparations may include conventional syringes and needles, or devices designed for the ballistic delivery of solid vaccines (WO 99/27961), or transdermal patches (WO 97/48440, WO 98/28037) ; or are applied to the surface of the skin (transdermal or transcutaneous delivery, WO 98/20734, WO 98/28037).

When the vaccines of the present invention are to be administered to the skin, or more specifically to the dermis, the vaccine is in a low liquid volume, particularly a volume of between about 0.5 ml and 0.2 ml.

The content of antigens in skin or intradermal vaccines of the present invention may be similar to the conventional doses found in intramuscular vaccines (see above). However, a characteristic of skin or intradermal vaccines that the formulations may have a "low dose". Accordingly, protein antigens in "low dose" vaccines are optionally presented as being only 0.1 to 10 μg or 0.1 to 5 μg per dose; and the saccharide antigens (optionally conjugated) may be present in the range of 0.01-1 jg, or between 0.01 to 0.5 jg of saccharide per dose.

As used herein, the term "intradermal delivery" means the delivery of a vaccine in the region of the dermis to the skin. However, the vaccine is not necessarily located exclusively in the dermis. The dermis is the layer of the skin that is located between about 1.0 and about 2.0 mm from the surface on human skin, but there is some variation in amount between individuals and in different parts of the body. In general, the dermis can be expected to reach 1.5 mm below the surface of the skin. The dermis is located between the stratum corneum and the epidermis on the surface and the subcutaneous layer below. Depending on the mode of delivery, the vaccine may ultimately be located only or primarily in the dermis, or may ultimately spread to the epidermis and dermis.

The present invention also provides an improved vaccine for the prevention or improvement of otitis media produced by Haemophilus influenzae by the addition of Haemophilus influenzae proteins , for example protein D in free or conjugated form. In addition, the present invention additionally provides an improved vaccine for the prevention or amelioration of pneumococcal infection in infants (eg, otitis media), based on the addition of one or more pneumococcal proteins such as free or conjugated proteins of the conjugated compositions of S pneumoniae of the invention. Said free pneumococcal proteins may be the same or different from any of the S. pneumoniae proteins that are used as protein carriers. One or more Moraxella catarrhalis protein antigens can also be included in the vaccine combination in a free or conjugated form. Therefore, the present invention is an improved method to give rise to an immune (protective) response against otitis media in infants.

In another embodiment, the present invention is an improved method for giving rise to an immune (protective) response in infants (defined as 0-2 years in the context of the present invention) by administering an effective and safe amount of a vaccine from the invention [a pediatric vaccine]. Other embodiments of the present invention include the provision of compositions of antigenic conjugates of S. pneumoniae of the invention for use in medicine and the use of conjugates of S. pneumoniae of the invention in the manufacture of a medicament for the prevention (or treatment ) of pneumococcal disease.

In yet another embodiment, the present invention is an improved method for giving rise to an immune (protective) response in an elderly population (in the context of the present invention a patient is considered elderly if he is 50 years of age or older, usually by over 55 years and more generally above 60 years) administering an effective and safe amount of the vaccine of the invention, optionally in conjunction with one or two proteins of S. pneumoniae present as free or conjugated protein, whose proteins of S Free pneumoniae may be the same or different from the proteins of S. pneumoniae that are used as protein vehicles.

A further aspect of the invention is a method for immunizing a human host against a disease caused by S. pneumoniae and optionally Haemophilus influenzae infection comprising the administration to the host of an immunoprotective dose of the immunogenic composition or vaccine or kit of the invention.

A further aspect of the invention is an immunogenic composition of the invention for use in the treatment or prevention of disease caused by infection with S. pneumoniae and optionally Haemophilus influenzae.

A further aspect of the invention is the use of the immunogenic composition or vaccine or kit of the invention in the manufacture of a medicament for the treatment or prevention of diseases caused by infection with S. pneumoniae and optionally Haemophilus influenzae.

In a further aspect, the disease is either pneumonia or invasive pneumococcal disease (IPD) of elderly humans, exacerbations of chronic obstructive pulmonary disease (COPD) of elderly humans, meningitis and / or bacteremia of childhood humans, otitis media of childhood human beings or pneumonia and / or conjunctivitis of childhood human beings.

The terms "comprising", "comprising" and "comprising" in the present document are intended by the inventors to be optionally substituted with the expressions "consisting of", "consisting of" and "consisting of", respectively , in each case.

Embodiments herein referring to "vaccine compositions" of the invention may also be applied to embodiments that refer to "immunogenic compositions" of the invention and vice versa.

The embodiments of the invention are further described in the subsequent numbered paragraphs:

Paragraph 1. An immunogenic composition comprising conjugates of S. pneumoniae capsular saccharide of serotypes 19A and 19F in which 19A is conjugated with a first bacterial toxoid that is pneumolysin, diphtheria toxoid or CRM197 and 19F is conjugated with a second bacterial toxoid which is diphtheria toxoid or CRM197 and which additionally comprises conjugates of the capsular saccharides of S. pneumoniae 4, 6B, 9V, 14, 18C, 23F, 1, 5 and 7F, in which the average size of the saccharide 19A is above 100 kDa

Paragraph 2. The immunogenic composition of paragraph 1, wherein the first bacterial toxoid is a protein different from the second bacterial toxin.

Paragraph 3. The immunogenic composition of any one of paragraphs 1-2 in which the first bacterial toxoid is pneumolysin.

Paragraph 4. The immunogenic composition of any one of paragraphs 1-3 in which the second bacterial toxoid is diphtheria toxoid.

Paragraph 5. The immunogenic composition of paragraphs 1-4 further comprising a capsule saccharide conjugate of S. pneumoniae 22F.

Paragraph 6. The immunogenic composition of paragraphs 1-5 which additionally comprises a capsule saccharide conjugate of S. pneumoniae 3.

Paragraph 7. The immunogenic composition of paragraphs 1-6 further comprising a capsule saccharide conjugate of S. pneumoniae 6A.

Paragraph 8. The immunogenic composition of any one of paragraphs 1-7 wherein 2 different carrier proteins are separately conjugated with at least 2 different serotypes of capsular saccharides of S. pneumoniae.

Paragraph 9. The immunogenic composition of any one of paragraphs 5-7 wherein 3 different carrier proteins are conjugated separately with at least 3 different serotypes of capsular saccharides of S. pneumoniae.

Paragraph 10. The immunogenic composition of any one of paragraphs 5-7 wherein 4 different carrier proteins are conjugated separately with at least 4 different serotypes of capsular saccharides of S. pneumoniae.

Paragraph 11. The immunogenic composition of any one of paragraphs 5-7 wherein 5 different carrier proteins are conjugated separately with at least 5 different serotypes of capsular saccharides of S. pneumoniae.

Paragraph 12. The immunogenic composition of paragraph 11 comprising 2 or more of the carrier proteins selected from the following list: tetanus toxoid, diphtheria toxoid, pneumolysin, protein D and PhtD or fusion proteins thereof.

Paragraph 13. The immunogenic composition of paragraphs 1-12 comprising the capsular saccharide of S. pneumoniae 1 conjugated to protein D.

Paragraph 14. The immunogenic composition of paragraphs 1-13 comprising the capsular saccharide of S. pneumoniae 3 conjugated to protein D, pneumolysin, or PhtD or fusion protein thereof.

Paragraph 15. The immunogenic composition of paragraphs 1-14 comprising the capsular saccharide of S. pneumoniae 4 conjugated to protein D.

Paragraph 16. The immunogenic composition of paragraphs 1-15 comprising the capsular saccharide of S. pneumoniae conjugated to protein D.

Paragraph 17. The immunogenic composition of paragraphs 1-16 comprising the capsular saccharide of S. pneumoniae 6B conjugated to protein D.

Paragraph 18. The immunogenic composition of paragraphs 1-17 comprising the capsular saccharide of S. pneumoniae 7F conjugated with protein D.

Paragraph 19. The immunogenic composition of paragraphs 1-18 comprising the capsular saccharide of S. pneumoniae 9V conjugated to protein D.

Paragraph 20. The immunogenic composition of paragraphs 1-19 comprising the capsular saccharide of S. pneumoniae 14 conjugated to protein D.

Paragraph 21. The immunogenic composition of paragraphs 1-20 which comprises the capsular saccharide of S. pneumoniae 23F conjugated with protein D.

Paragraph 22. The immunogenic composition of paragraphs 1-21 comprising the capsular saccharide of S. pneumoniae 18C conjugated to tetanus toxoid.

Paragraph 23. The immunogenic composition of paragraphs 1-22 comprising the capsular saccharide of S. pneumoniae 19A conjugated with pneumolysin.

Paragraph 24. The immunogenic composition of paragraphs 1-23 comprising the capsular saccharide of S. pneumoniae 22F conjugated to PhtD or fusion protein thereof.

Paragraph 25. The immunogenic composition of paragraphs 1-24 comprising the capsular saccharide of S. pneumoniae 6A conjugated to pneumolysin or a protein of H. influenzae, optionally protein D or PhtD or fusion protein thereof.

Paragraph 26. The immunogenic composition according to any preceding paragraph wherein the capsular saccharide 19A is directly conjugated to the carrier protein.

Paragraph 27. The immunogenic composition of any one of paragraphs 1-25 wherein the capsular saccharide 19A is conjugated to the carrier protein through a linker.

Paragraph 28. The immunogenic composition of paragraph 27 in which the linker is ADH.

Paragraph 29. The immunogenic composition of paragraph 27 or 28 wherein the linker is bound to the carrier protein by the carbodiimide chemistry, optionally using EDAC.

Paragraph 30. The immunogenic composition of any one of paragraphs 26-29 wherein the saccharide 19A is conjugated to the carrier protein or linker using the CDAP chemistry.

Paragraph 31. The immunogenic composition according to any of paragraphs 1-26 wherein the ratio of vehicle protein to saccharide 19F is between 5: 1 and 1: 5, 4: 1 and 1: 1 or 3.5 : 1 and 2.5: 1 (w / w). Paragraph 32. The immunogenic composition according to any of the preceding paragraphs wherein the 19F capsular saccharide is directly conjugated to the carrier protein.

Paragraph 33. The immunogenic composition of any one of paragraphs 1-31 wherein the 19F capsular saccharide is conjugated to the vehicle protein through a linker.

Para 34. The immunogenic composition of paragraph 33 in which the linker is ADH.

Paragraph 35. The immunogenic composition of paragraph 33 or 34 wherein the linker is bound to the carrier protein by the carbodiimide chemistry, optionally using EDAC.

Paragraph 36. The immunogenic composition of any one of paragraphs 32-35 wherein the 19F saccharide is conjugated to the carrier protein or linker using the CDAP chemistry.

Paragraph 37. The immunogenic composition of any one of paragraphs 1-36 wherein the ratio of vehicle protein to saccharide 19F is between 5: 1 and 1: 5, 4: 1 and 1: 1 or 2: 1 and 1: 1 (p / p).

Paragraph 38. The immunogenic composition of any one of paragraphs 1-37 comprising a 22F capsular saccharide directly conjugated to the carrier protein.

Paragraph 39. The immunogenic composition of any one of paragraphs 1-37 comprising a 22F capsular saccharide conjugated to the carrier protein via a linker.

Paragraph 40. The immunogenic composition of paragraph 39 wherein the linker is ADH.

Paragraph 41. The immunogenic composition of paragraph 39 or 40, wherein the linker is bound to the carrier protein by carbodiimide chemistry, optionally using EDAC.

Paragraph 42. The immunogenic composition of any one of paragraphs 38-41, wherein the saccharide 22F is conjugated to the carrier protein or linker using the CDAP chemistry.

Paragraph 43. The immunogenic composition of any one of paragraphs 1-42, wherein the ratio of vehicle protein to saccharide 22F is between 5: 1 and 1: 5, 4: 1 and 1: 1 or 2: 1 and 1: 1 (p / p).

Paragraph 44. The immunogenic composition of any of the preceding paragraphs wherein the average size of saccharide 19A is between 110 and 700 kDa, 110-300, 120-200, 130-180, or 140-160 kDa.

Paragraph 45. The immunogenic composition of paragraph 44 wherein the saccharide 19A is either a natural polysaccharide or is sized by a factor of not more than x5.

Paragraph 46. The immunogenic composition of paragraph 44 or 45 wherein the saccharide 19A has been sized by microfluidization.

Paragraph 47. The immunogenic composition of any of the preceding paragraphs wherein the dose of the saccharide conjugate 19A is between 1 and 10 mg, 1 and 5 mg or 1 and 3 mg of saccharide.

Paragraph 48. The immunogenic composition of paragraph 47 wherein the dose of the saccharide conjugate 19A is 3 mg of saccharide.

Paragraph 49. The immunogenic composition of any preceding paragraph comprising a saccharide conjugate 22F wherein the average size of saccharide 22F is above 100 kDa.

Paragraph 50. The immunogenic composition of paragraph 49, wherein the average size of saccharide 22F is between 110 and 700 kDa, 110-300, 120-200, 130-180 or 150-170 kDa.

Paragraph 51. The immunogenic composition of paragraph 49 or 50 wherein the saccharide 22F is either a natural polysaccharide or is dimensioned by a factor of not more than x5.

Paragraph 52. The immunogenic composition of paragraph 49, 50051 in which saccharide 22F has been sized by microfluidization.

Paragraph 53. The immunogenic composition of any preceding paragraph comprising a saccharide conjugate 22F, wherein the dose of the saccharide conjugate 22F is between 1 and 10 mg, 1 and 5 mg, or 1 and 3 mg of saccharide.

Paragraph 54. The immunogenic composition of paragraph 53 wherein the dose of saccharide conjugate 22F is 3 mg of saccharide.

Paragraph 55. The immunogenic composition of any preceding paragraph in which the average saccharide size is above 50 kDa.

Paragraph 56. The immunogenic composition according to paragraph 55, comprising serotype 1 having an average saccharide size of between 300 and 400 kDa.

Paragraph 57. The immunogenic composition according to paragraph 55 or 56 comprising serotype 4 having an average saccharide size of between 75 and 125 kDa.

Paragraph 58. The immunogenic composition according to paragraph 55, 56 or 57 comprising serotype 5 having an average saccharide size of between 350 and 450 kDa.

Paragraph 59. The immunogenic composition according to any of paragraphs 55 to 58 comprising serotype 6B having an average saccharide size of between 1000 and 1400 kDa.

Paragraph 60. The immunogenic composition according to any of paragraphs 55 to 59 comprising serotype 7F having an average saccharide size of between 200 and 300 kDa.

Paragraph 61. The immunogenic composition according to any of paragraphs 55 to 60 comprising serotype 9V having an average saccharide size of between 250 and 300 kDa.

Paragraph 62. The immunogenic composition according to any of paragraphs 55 to 61 comprising serotype 14 having an average saccharide size of between 200 and 250 kDa.

Paragraph 63. The immunogenic composition according to any of paragraphs 55 to 62 comprising serotype 23F having an average saccharide size of between 900 and 1000 kDa.

Paragraph 64. The immunogenic composition of any of the preceding paragraphs comprising serotypes 5, 6B and 23F (and optionally 6A) as natural saccharides.

Paragraph 65. The immunogenic composition of any of the preceding paragraphs wherein the dose of the capsular saccharide conjugates is between 1 and 10 mg, 1 and 5 mg or 1 and 3 mg of saccharide per conjugate.

Paragraph 66. The immunogenic composition of any of the preceding paragraphs comprising the conjugates of serotypes 4, 18C, 19F and 22F (and optionally 19A) in dosages of 3 mg of saccharide per conjugate.

Paragraph 67. The immunogenic composition of any preceding paragraph comprising the conjugates of serotypes 1, 5, 6B, 7F, 9V, 14 and 23F (and optionally 6A and / or 3) in dosages of 1 mg of saccharide per conjugate.

Paragraph 68. The immunogenic composition of any of the preceding paragraphs additionally comprising S. pneumoniae saccharides of the serotypes other than the conjugates, such that the number of conjugated and non-conjugated saccharide serotypes is less than or equal to 23.

Paragraph 69. The immunogenic composition of any of the preceding paragraphs further comprising one or more conjugated or unconjugated proteins of S. pneumoniae.

Paragraph 70. The immunogenic composition of paragraph 69, which comprises one or more non-conjugated proteins of S. pneumoniae.

Paragraph 71. The immunogenic composition of paragraph 69 or 70 wherein said one or more proteins of S. pneumoniae are selected from the family of the polyhistidine triad (PhtX), the family of proteins that bind to choline (CbpX), Truncated CbpX, the family of LytX, truncated LytX, truncated CbpX-LytXtruncada chimeric proteins, detoxified pneumolysin (Ply), PspA, PsaA, Sp128, Sp101, Sp130, Sp125 and Sp133.

Para 72. The immunogenic composition of paragraphs 69, 70 or 71 comprising pneumolysin.

Paragraph 73. The immunogenic composition of any of paragraphs 69 to 72 comprising a PhtX protein.

Paragraph 74. The immunogenic composition according to any preceding paragraph comprising pneumolysin as a free or vehicle protein.

Paragraph 75. The immunogenic composition according to any preceding paragraph comprising a PhtX protein as a free or vehicle protein.

Paragraph 76. The immunogenic composition of paragraph 75 wherein said PhtX protein is PhtD or a PhtBD or a PhtDE fusion protein.

Paragraph 77. The immunogenic composition according to any preceding paragraph additionally comprising an adjuvant.

Paragraph 78. The immunogenic composition of paragraph 77, wherein the adjuvant comprises a liposome vehicle.

Paragraph 79. The immunogenic composition of paragraph 78, wherein the adjuvant comprises (per 0.5 ml dose) 0.1-10 mg, 0.2-7, 0.3-5, 0.4-2 or 0.5-1 mg (eg, 0.4-0.6, 0.9-1.1, 0.5 or 1 mg) phospholipid (eg, DOPC).

Paragraph 80. The immunogenic composition of paragraph 78 or 79, wherein the adjuvant comprises (per dose of 0.5 ml) 0.025-2.5, 0.05-1.5, 0.075-0.75, 0.1 -0.3, or 0.125-0.25 mg (eg, 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg) of sterol (eg, cholesterol).

Paragraph 81. The immunogenic composition of paragraphs 78-80, wherein the adjuvant comprises (per 0.5 ml dose) 5-60, 10-50 or 20-30 mg (eg, 5-15, 40- 50, 10, 20, 30, 40 or 50 mg) of lipid A derivative (eg, 3d-MPL).

Paragraph 82. The immunogenic composition of paragraphs 78-81, wherein the adjuvant comprises (per 0.5 ml dose) 5-60, 10-50 or 20-30 mg (eg, 5-15, 40- 50, 10, 20, 30, 40 or 50 mg) of saponin (for example QS21).

Paragraph 83. The immunogenic composition of paragraph 77, wherein the adjuvant comprises an oil in water emulsion.

Paragraph 84. The immunogenic composition of paragraph 83, wherein the adjuvant comprises (per 0.5 ml dose) 0.5-15, 1-13, 2-11, 4-8, or 5-6 mg (per example, 2-3, 5-6, or 10-11 mg) of metabolizable oil (such as squalene) ..

Paragraph 85. The immunogenic composition of paragraph 83 or 84, wherein the adjuvant comprises (per dose of 0.5 ml) 0.1-10, 0.3-8, 0.6-6, 0.9-5 , 1-4 or 2-3 mg (eg, 0.9-1.1, 2-3 or 4-5 mg) of emulsifier (such as Tween 80).

Paragraph 86. The immunogenic composition of paragraphs 83-85, in which the adjuvant comprises (per 0.5 ml dose) 0.5-20, 1-15, 2-12, 4-10, 5-7 mg (for example 11-13, 5-6, or 2-3 mg) of tocol (such as alpha tocopherol).

Paragraph 87. The immunogenic composition of paragraphs 83-86 wherein the adjuvant comprises (per 0.5 ml dose) 5-60, 10-50 or 20-30 mg (e.g., 5-15, 40-50 , 10, 20, 30, 40 or 50 mg) of lipid derivative A (e.g. 3D-MPL).

Paragraph 88. The immunogenic composition of paragraphs 83-87, wherein the adjuvant comprises (per 0.5 ml dose) 0.025-2.5, 0.05-1.5, 0.075-0.75, 0, 1-0.3 or 0.125-0.25 mg (eg, 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg) of sterol (eg, cholesterol).

Paragraph 89. The immunogenic composition of paragraphs 83-88, wherein the adjuvant comprises (per 0.5 ml dose) 5-60, 10-50 or 20-30 mg (eg, 5-15, 40- 50, 10, 20, 30, 40 or 50 mg) of saponin (e.g., QS21).

Paragraph 90. The immunogenic composition of paragraph 77, wherein the adjuvant comprises a metal salt and a lipid derivative A.

Paragraph 91. The immunogenic composition of paragraph 90, wherein the adjuvant comprises (per dose of 0.5 ml) 100-750, 200-500 or 300-400 mg of Al as aluminum phosphate.

Paragraph 92. The immunogenic composition of paragraph 90 or 91, wherein the adjuvant comprises (per dose of 0.5 ml) 5-60, 10-50 or 20-30 mg (eg, 5-15, 40-50) , 10, 20, 30, 40 or 50 mg) of lipid A derivative (e.g., 3D-MPL).

Paragraph 93. The immunogenic composition of any one of paragraphs 1-92, which comprises at least or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 13 capsular saccharides of conjugated S. pneumoniae with PhtD or fusion protein of it.

Paragraph 94. The immunogenic composition of any one of paragraphs 1-92, which comprises at least or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 13 capsular saccharides of conjugated S. pneumoniae with pneumolysin.

Paragraph 95. A vaccine kit comprising an immunogenic composition according to any one of paragraphs 1 to 92 and further comprising for the simultaneous or sequential administration an adjuvant as defined in any of paragraphs 78 to 94.

Paragraph 96. A vaccine comprising the immunogenic composition of any one of paragraphs 1-94 and a pharmaceutically acceptable excipient.

Paragraph 97. A method for preparing the vaccine according to paragraph 96 comprising the step of mixing the immunogenic composition of any one of paragraphs 1 to 94 with a pharmaceutically acceptable excipient.

Para 98. A method of immunizing a human host against the disease caused by Streptpcoccus pneumoniae infection comprising administering to the host an immunoprotective dose of the immunogenic composition of any one of paragraphs 1-94 or the vaccine of paragraph 96.

Paragraph 99. The procedure of paragraph 98, in which the human host is elderly, and the disease is either or both of pneumonia or invasive pneumococcal disease (IPD).

Paragraph 100. The procedure of paragraph 98 or 99, in which the human host is an elderly person, and the disease is exacerbations of chronic obstructive pulmonary disease (COPD).

Paragraph 101. The procedure of paragraph 98, in which the human host is an infant and the disease is otitis media.

Paragraph 102. The procedure of paragraph 98 or 101, in which the human host is an infant, and the disease is meningitis and / or bacteremia.

Paragraph 103. The procedure of paragraphs 98, 101 or 102, in which the human host is an infant, and the disease is pneumonia and / or conjunctivitis.

Paragraph 104. The immunogenic composition of paragraphs 1-94 or the vaccine of paragraph 96 for use in the treatment of the prevention of disease caused by Streptococcus pneumoniae infection .

Paragraph 105. The use of the immunogenic composition or vaccine of paragraphs 1-94 or the vaccine of paragraph 96 in the manufacture of a medicament for the treatment or prevention of diseases caused by Streptococcus pneumoniae infection .

Paragraph 106. The use of paragraph 105, in which the disease is either or both of pneumonia or invasive pneumococcal disease (IPD) in elderly humans.

Paragraph 107. The use of paragraph 105 or 106, in which the disease is exacerbations of chronic obstructive pulmonary disease (COPD) of elderly humans.

Paragraph 108. The use of paragraph 105, in which the disease is otitis media of lactating human beings.

Paragraph 109. The use of paragraph 105 or 108, in which the disease is meningitis and / or bacteremia of lactating human beings.

Paragraph 110. The use of paragraphs 105, 108 or 109, in which the disease is pneumonia and / or conjunctivitis of lactating humans.

In order that the present invention be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not intended to limit the scope of the invention in any way. Examples

Example 1: Expression of Protein D

Protein D of Haemophilus influenzae

Genetic construction for protein D expression

Starting materials

The DNA encoding protein D

Protein D is highly conserved among all serotypes and non-typable strains of H. influenzae. The vector pHIC348 containing the DNA sequence encoding the complete Protein D gene was obtained by Dr. A. Forsgren, Department of Medical Microbiology, University of Lund, Malmo General Hospital, Malmo, Sweden. The DNA sequence of protein D has been published by Janson et al., (1991) Infect. Immun. 59: 119-125.

Expression vector pMG1

The expression vector pMG1 is a derivative of pBR322 (Gross et al., 1985) in which control elements derived from bacteriophage A were introduced for the transcription and translation of inserted foreign genes (Shatzman et al., 1983). In addition, the ampicillin resistance gene was exchanged for the kanamycin resistance gene.

The strain of E. coli AR58

The E. coli strain AR58 was generated by transduction of N99 with a P1 phage raw material previously cultured in a derivative SA500 (gaIE :: TN10, lambdakil-cl857 AH1). N99 and SA500 are E. coli K12 strains derived from Dr. Martin Rosenberg's laboratory at the National Institute of Health.

The expression vector pMG1

For the production of protein D, the DNA encoding the protein was cloned into the expression vector pMG1. This plasmid uses signals from lambda phage DNA to direct the transcription and translation of inserted foreign genes. The vector contains the PL promoter, OL operator and two sites of use (NutL and NutR) to alleviate the effects of transcriptional polarity when the N protein is provided (Gross et al., 1985). The vectors containing the PL promoter are introduced into a lysogenic E. coli host to stabilize the plasmid DNA. Lysogenic host strains contain replication-deficient lambda phage DNA integrated into the genome (Shatzman et al., 1983). The chromosomal lambda phage DNA directs the synthesis of the repressor protein cl that binds to the OL repressor of the vector and prevents the binding of the RNA polymerase to the PL promoter and in this way the transcription of the inserted gene. The cl gene of expression strain AR58 contains a temperature-sensitive mutant so that PL-directed transcription can be regulated by a change in temperature, i.e., an increase in culture temperature inactivates the repressor and the synthesis of the foreign protein starts. This expression system allows controlled synthesis of foreign proteins, especially those that can be toxic to the cell (Shimataka and Rosenberg, 1981).

The AR58 strain of E. coli

The E. coli lysogenic strain E.sub.58 which is used for the production of the protein carrier D is a derivative of the N99 strain of E. coli K12 NIH (F- su- galK2, lacZ-thr-). It contains a deficient lysogenic lambda phage (galE :: TN10, lambdakil-cl857 AH1). The Kil- phenotype prevents the silencing of the macromolecular synthesis of the host. The cI857 mutation confers a temperature-sensitive lesion of the cl repressor. The AH1 elimination removes the right operon from phage lambda and the host loci bio, uvr3, and chlA. Strain AR58 was generated by transduction of N99 with a phage raw material P1 previously cultivated in a derivative of SA500 (galE :: TN10, lambdaKil- cl857 AH1). The introduction of N99-deficient lysogene was selected with tetracycline thanks to the presence of a TN10 transposon that encodes tetracycline resistance in the adjacent galE gene.

Construction of the vector pMGMDPPrD

The pMG1 vector containing the gene encoding the non-structural S1 protein of influenza virus (pMGNS1) was used to construct pMGMDPPrD. The protein D gene was amplified by PCR from the vector pHIC348 (Janson et al., 1991 Infect. Immun 59: 119-125) with the PCR primers containing the Ncol and Xbal restriction sites at the ends of the protein. 'and 3', respectively. The Ncol / Xbal fragment was then introduced into the pMGNS1 between Ncol and Xbal thus creating a fusion protein containing the 81 amino acids of the N-terminus of the NS1 protein followed by the PD protein. This vector was labeled pMGNS1PrD.

Based on the construction described above, the final construct for the expression of protein D was generated. A BamHI / BamHI fragment was removed from pMGNS1PrD. This DNA hydrolysis removes the NS1 coding region, except for the first three three residues of the N-terminus. By re-joining in the vector a gene encoding a fusion protein with the following N-terminal amino acid sequence was generated:

- MDHS SSHSSNMANT --- NS1 Protein D

Protein D contains no leader peptide or cysteine at the N-terminus to which lipid chains are normally fixed. The protein is therefore neither excreted in the periplasm nor lipidated, and remains in the cytoplasm in soluble form.

The final construct pMG-MDPPrD was introduced into host strain AR58 by thermal shock at 37 ° C. The bacteria containing the plasmid were selected in the presence of kanamycin. The presence of the DNA insert encoding protein D was demonstrated by digestion of isolated plasmid DNA with selected endonucleases. Reference is made to the recombinant strain of E. coli as ECD4.

The expression of protein D is under the control of the lambda PL promoter / OL operator. Host strain AR58 contains a cl gene sensitive to temperature in the genome which blocks the expression of PL lambda at low temperature by binding to OL. Once the temperature is raised, the Cl of Ol is released and protein D is expressed.

Small scale preparation

At the end of the fermentation the cells were concentrated and frozen.

The extraction of harvested cells and the purification of protein D was carried out in the following manner. The agglomerate of the frozen cell culture was thawed and re-suspended in a cell destruction solution (citrate buffer pH 6.0) at an OD 650 = 60 final. The suspension was passed twice through a high pressure homogenizer at P = 1000 bar. The cell culture homogenate was clarified by centrifugation and the cell debris was removed by filtration. In the first purification step the filtered lysate is applied to a cation exchange chromatography column (SP Sepharose Fast Flow). The PD binds to the gel matrix by ionic interaction and is eluted by a step of increasing the ionic strength of the elution buffer.

In a second purification step impurities are retained in an anion exchange matrix (Q Sepharose fast flow). The PD does not bind in the gel and can be collected in the continuous flow.

In both steps of chromatography columns the collected fraction is controlled by DO. The continuous flow of the anion exchange column chromatography containing the purified protein D is concentrated by ultrafiltration.

Protein D containing the retentate of the ultrafiltration is finally passed through a 0.2 mm membrane.

Large-scale preparation

The extraction of the harvested cells and the purification of protein D was carried out in the following manner. The collected broth was cooled and passed directly twice through a high pressure homogenizer at a pressure of about 800 bar.

In the first purification step the cell culture homogenate is diluted and applied to a cation exchange chromatography column (SP Sepharose of large beads). The PD binds to the gel matrix by ionic interaction and is eluted by a step of increasing the ionic strength of the elution buffer and filtered.

In a second purification step impurities are retained in an anion exchange matrix (Q Sepharose fast flow). The PD does not bind to the gel and can be collected in the continuous flow.

In both steps of column chromatography the collection is controlled by DO. The continuous flow of the anion exchange column chromatography containing the purified protein D is concentrated and diafiltered by ultrafiltration.

Protein D contained in the ultrafiltration retentate is finally passed through a 0.2 mm membrane.

Example 1 b: Expression of PhtD

The PhtD protein is a member of the pneumococcal histidine (Pht) triad protein family that is characterized by histidine triads (motif HXXHXHX). PhtD is a molecule of 838 aa and has 5 triads of histidine (see Medlmmune WO00 / 37105 SEQ ID NO: 4 for the amino acid sequence and SEQ ID NO: 5 for the DNA sequence). PhtD also contains a proline-rich region in the middle (amino acid positions 348-380). The PhtD has a signal sequence at the N-terminus of 20 a with a LXXC motif.

Genetic construction

The genetic sequence of the PhDD MedImmune protein (from aa 21 to aa 838) was recombinantly transferred to E. coli using a pTCMP14 domestic vector carrying the pA promoter. The E. coli host strain is AR58, which harbors the thermosensitive repressor cI857, which allows heat induction of the promoter.

The polymerase chain reaction was carried out to amplify the phtD gene from the Medlmmune plasmid (which harbors the phtD gene of Norway strain 4 of Streptococcus pneumoniae (serotype 4) - SEQ ID NO: 5 as described in FIG. WO 00/37105). The primers, specific only for the phtD gene, were used to amplify the phtD gene in two fragments. The primers had each restriction sites NdeI and KpnI and KpnI or Xbal. These primers do not hybridize to any nucleotide in the vector but only to the specific sequences of the phtD gene . An artificial ATG start codon was inserted using the first primer having the Ndel restriction site. The generated PCR products were then inserted into the cloning vector pGEM-T (Promega), and the DNA sequence was confirmed. The subcloning of the fragments was then carried out in the TCMP14 expression vector using conventional techniques and the vector transformed into E. coli AR58.

Purification of PhtD

The purification of PhtD was achieved in the following way:

□ Culture of E. coli cells in the presence of kanamycin: culture for 30 hours at 30 ° C, then induction for 18 hours at 39.5 ° C.

□ Destruction of E. coli cells from the whole culture at an OD of 6115 in the presence of 5 mM EDTA and 2 mM PMSF as protease inhibitors: Rannie, 2 passages, 1000 bar.

□ Capture of antigens and elimination of cellular debris on XL Q Streamline chromatography in expanded bed mode at room temperature (20 ° C); the column was washed with 150 mM NaCl Empigen 0.25% pH 6.5 and eluted with 400 mM NaCl Empigen 0.25% in 25 mM potassium phosphate buffer pH 7.4.

□ Filtration in a Sartobran 150 cartridge (0.45 0.2 mm)

□ Antigen binding with chromatography on Sepharose FF IMAC Chelating Zn ++ at pH 7.4 in the presence of 5 mM imidazole at 4 ° C; the column is washed with 5 mM imidazole and 1% Empigen and eluted with 50 mM imidazole, both in 25 mM potassium phosphate buffer pH 8.0.

□ Weak anion exchange chromatography in positive mode on Fractogel EMD DEAE at pH 8.0 (25 mM potassium phosphate) at 4 ° C; the column was washed with 140 mM NaCl and eluted at 200 mM NaCl while the contaminants (proteins and DNA) were kept adsorbed in the exchanger.

□ Concentration and ultrafiltration with 2 mM Na / phosphate K pH 7.15 on a 50 kDa membrane.

□ Sterilization by filtration of the purified volume in a Millipak-200.2 mm filter cartridge.

Example 1c: Expression of pneumolysin

Pneumococcal pneumolysin was prepared and detoxified as described in WO2004 / 081515 and WO2006 / 032499.

Example 2:

Preparation of conjugates

It is well known in the art how to make purified pneumococcal polysaccharides. For the purposes of these examples, the polysaccharides were produced essentially as described in EP072513 or by closely related procedures. Before conjugation the polysaccharides have to be modified in size by microfluidization as described below.

Activation and coupling conditions are specific to each polysaccharide. These are given in Table 1. The modified polysaccharide size (except pS5, 6B and 23F) was dissolved in 2M NaCl, 0.2M NaCl or in water for injection (WFI). The optimal concentration of polysaccharide was evaluated for all serotypes. All serotypes except serotype 18C were conjugated directly to the protein carrier as detailed below. Two alternative conjugates were made with serotype 22F; one conjugate directly, the other by means of an ADH linker.

From a raw material solution of 100 mg / ml in acetonitrile or in a 50% / 50% acetonitrile / water solution, CDAP (ratio CDAP / PS 0.5-1.5 mg / mg PS) was added to the polysaccharide solution. 1.5 minutes later, 0.2 M-0.3 M NaOH was added to obtain the specific activation pH. Activation of the polysaccharide was carried out at this pH for 3 minutes at 25 ° C. The purified protein (protein D, PhtD, pneumolysin or DT) (the amount depends on the initial PS / protein carrier ratio) was added to the activated polysaccharide and the specific pH coupling reaction was carried out for up to 2 hours (depending on the serotype) under regulation the pH. In order to inactivate the non-reactive cyanate ester groups, a solution of 2 M glycine was then added to the mixture. The pH was then adjusted to the inactivation pH (pH 9.0). The solution was stirred for 30 minutes at 25 ° C and then overnight at 2-8 ° C with slow continuous stirring.

Preparation of 18C:

18C was bound to the protein vehicle by means of a dihydrazide adipic acid (ADH) linker. The serotype of polysaccharide 18C was microfluidized before conjugation.

Derivation of tetanus toxoid with EDAC

For the derivation of the tetanus toxoid, the purified TT was diluted to 25 mg / ml in 0.2 M NaCl and the ADH spacer was added in order to reach a final concentration of 0.2 M. When the solution of the spacer, the pH was adjusted to 6.2. EDAC (1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide) was then added until a final concentration of 0.02 M was reached and the mixture was stirred for 1 hour under the regulating pH. The condensation reaction was stopped by increasing the pH to 9.0 for at least 30 minutes at 25 ° C. The derivatized TT was then diafiltered (10 kDa CO membrane) in order to remove the residual ADH and the EDAC reagent.

The mass TTah was finally sterilized by filtration to the coupling stage and stored at -70 ° C.

TT chemical coupling a h to PS 18C

The details of the conjugation parameters can be found in Table 1.

2 grams of microfluidized PS were diluted to the defined concentration in water and adjusted to 2 M NaCl by the addition of NaCl powder.

The CDAP solution (100 mg / ml freshly prepared acetonitrile / WFI 50/50 v / v) was added until the appropriate CDAP / PS ratio was reached.

The pH was raised to the activation pH pH 9.0 by the addition of 0.3 M NaOH and stabilized at this pH until the addition of TT ah .

After 3 minutes, the derivatized TT ah (20 mg / ml in 0.2 M NaCl) was added to each ratio TT ah / PS of 2; the pH was regulated at coupling pH 9.0. The solution was left for one hour under the regulation pH.

For inactivation, a 2 M glycine solution was added to the PS / TT mixture ah / CDAP.

The pH was adjusted to inactivation pH (pH 9.0).

The solution was stirred for 30 min at 25 ° C, and then left overnight at 2-8 ° C with slow continuous stirring. Conjugate PS22FAH-PhtD

In a second conjugation procedure for this saccharide (the first was the direct conjugation procedure PS22-PhtD shown in Table 1), 22F was bound to the protein carrier by means of a linker-adipic acid dihydrazide (ADH) . The serotype 22F polysaccharide was microfluidized before conjugation.

Derivation PS 22F

Activation and coupling were carried out at 25 ° C under continuous stirring in a water bath with temperature control.

The microfluidized PS22F was diluted to obtain a final PS concentration of 6 mg / ml in 0.2 M NaCl and the solution was adjusted to a pH of 6.05 ± 0.2 with 0.1 N HCl.

The CDAP solution (100 mg / ml acetonitrile / WFI, 50/50 freshly prepared) was added until reaching the appropriate CDAP / PS ratio (1.5 / 1 w / w).

The pH was raised to activation pH 9.00 ± 0.05 by the addition of 0.5 M NaOH and stabilized at this pH until the addition of ADH.

After 3 minutes, the ADH was added until reaching the appropriate ADH / PS ratio (8.9 / 1 w / w); the pH was adjusted to pH of coupling pH 9.0. The solution was left for 1 hour under regulation pH.

The PS derivative ah was concentrated and diafiltered.

Coupling

The PhtD at 10 mg / ml in 0.2 M NaCl was added to the PS22F derivative ah in order to achieve a PhtD / PS22FAH ratio of 4/1 (w / w). The pH was adjusted to 5.0 ± 0.05 with HCl. The EDAC solution (20 mg / ml in 0.1 M Tris-HCl pH 7.5) was added manually in 10 min (250 pl / min) to reach 1 mg EDAC / mg PS22F ah . The resulting solution was incubated for 150 min (also used for 60 min) at 25 ° C with stirring and pH regulation. The solution was neutralized by the addition of 1 M Tris-HCl pH 7.5 (1/10 of the final volume) and left 30 min at 25 ° C. Before elution in Sephacryl S400HR, the conjugate was clarified using a 5 mm Minisart filter.

The resulting conjugate has a final PhtD / PS ratio of 4.1 (w / w), a free PS content below 1% and an antigenicity (a-PS / a-PS) of 36.3% and anti-antigenicity -PhtD of 7.4%.

Purification of the conjugates:

The conjugates were purified by gel filtration using a Sephacryl S400HR gel filtration column equilibrated with 0.15 M NaCl (S500HR for 18C) to remove small molecules (including DMAP) and unconjugated PS and protein. Based on the different molecular sizes of the reaction components, the PS-PD, PS-TT, PS-PhtD, PS-pneumolysin or PS-DT conjugates are first eluted, followed by free PS or free DT and finally DMAP and other salts (NaCl, glycine).

The fractions containing the conjugates were detected by UV 280 nm. The fractions were grouped according to their Kd, sterilized by filtration (0.22 mm) and stored at 2-8 ° C. The relationships were determined PS / Protein in conjugate preparations.

Specific conditions of activation / coupling / inactivation of S. pneumoniae PS conjugates with Protein D / TT / DT / PhtD / Ply

When "pfluid" appears on a header line, it indicates that the saccharide was modified in size by microfluidization before conjugation. The sizes of saccharides after microfluidization are given in Table 2.

Figure imgf000031_0001

Characterization:

Each conjugate was characterized and fulfilled the specifications described in Table 2. The polysaccharide content (| jg / ml) was measured by the Resorcinol assay and the protein content (| jg / ml by the Lowry assay.) The Ps / ratio PD (w / w) is determined by the ratio of concentrations.

Content of free polysaccharide (%):

The free polysaccharide content of the conjugates which were kept at 4 ° C or stored days at 37 ° C in the supernatant obtained after incubation with anti-protein vehicle antibodies and saturated ammonium sulfate was determined, followed by centrifugation.

An a-PS / a-PS ELISA was used for the quantification of free polysaccharides in the supernatant. The absence of conjugate was also controlled by ELISA α-protein carrier / α-PS.

Antigenicity:

The antigenicity of the same conjugates was analyzed in a sandwich ELISA in which the capture and detection of antibody was a-PS and a-Protein, respectively.

Free protein content (%):

The unconjugated protein carrier can be separated from the conjugate during the purification step. The free residual protein content was determined using size exclusion chromatography (TSK 5000-PWXL) followed by UV detection (214 nm). The elution conditions allowed separation of the free protein carrier and the conjugate. The free protein content in the conjugate volumes was then determined against a calibration curve (from 0 to 560 jlg / μl of protein carrier). The free T-protein vehicle was obtained as follows:% free vehicle = (free vehicle (jg / ml) / (total concentration of the corresponding protein vehicle measured by Lowry (jg / ml) * 100%).

Stability:

The molecular weight distribution (Kav) and stability were measured by HPLC-SEC gel filtration (TSK 5000-PWXL) for conjugates that were kept at 4 ° C and stored for 7 days at 37 ° C.

The characterization of 10/11/13/14 valences is given in Table 2 (see comment below).

The protein conjugates can be adsorbed on aluminum phosphate and pooled to form the final vaccine.

Conclusion:

Immunogenic conjugates had been produced, which had been shown to be components of a promising vaccine.

TABLE 2 - Characteristics of the conjugates

Figure imgf000032_0001

(continuation)

Figure imgf000033_0001

A 10-valent vaccine was produced by mixing serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugates (for example at a dose of 1, 3, 1, 1, 1, 1, 1, 3, 3, 1 | jg of saccharide, respectively per dose for a human). An 11-valent vaccine was produced by also adding the conjugate of serotype 3 of Table 5 (for example, to 1 jg of saccharide per dose for a human being). A 13-valent vaccine was produced by also adding the conjugates of serotypes 19A and 22F above (with 22F bound either directly to PhtD, or alternatively by an ADH linker) [eg at a dose of 3 jg of each of the saccharides per dose for a human being]. A 14-valent vaccine was produced by also adding the conjugate of serotype 6A above [for example, at a dose of 1 jg saccharide per dose for a human].

Example 3: Evidence that the inclusion of Haemophilus influenzae protein D in an immunogenic composition of the invention may provide improved protection against acute otitis media (AOM)

Study design

The study used a 11Pn-PD vaccine - comprising serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F each conjugated with protein D of H. influenzae (which is made reference in Table 5 of Example 4). Subjects were randomized into two groups to receive four doses of either the 11 Pn-PD or Havrix vaccine at approximately 3, 4, 5 and 12-15 months of age. All subjects received the Infanrix-hexa vaccine from GSK Biologicals (DTPa-HBV-IPV / Hib) concomitantly at 3, 4 and 5 months of age. The Infanrix-hexa vaccine is a combination of Pediarix and Hib mixed before administration. The monitoring of the efficacy for the "According to the Protocol" analysis began 2 weeks after the administration of the third dose of vaccine and continued until 24-27 months of age. The nasopharyngeal transport of S. pneumoniae and H. influenzae was evaluated in a selected subgroup of subjects.

The parents were warned to consult the investigator if their child was sick, had an earache, spontaneous perforation of the tympanic membrane or spontaneous otic discharge. If the investigator suspected an episode of AOM, the child was immediately referred to an ear, nose and throat specialist (ENT) to confirm the diagnosis.

A clinical diagnosis of AOM was based on the visual appearance of the tympanic membrane (ie, redness, bulging, loss of light reflection) or the presence of middle ear fluid discharge (as demonstrated by simple or pneumatic otoscopy). or by microscopy). In addition, at least two of the following signs or symptoms had to be present: earache, ear discharge, hearing loss, fever, lethargy, irritability, anorexia, vomiting, or diarrhea. If the ENT specialist confirmed the clinical diagnosis, a specimen of the middle ear fluid was collected by tympanocentesis for the bacteriological test.

For subjects who repeated the sick visit, it was considered that a new AOM episode had started if more than 30 days had passed since the beginning of the previous episode. In addition, an episode of AOM was considered to be a new bacterial episode if the serotype of the isolated bacterium was different from the previous isolate whatever the interval between the two consecutive episodes.

Results of the trial

A total of 4968 children were enrolled, 2489 in the 11Pn-PD group and 2479 in the control group. There were no significant differences in the demographic characteristics or risk factors between the two groups.

Clinical episodes and definition of AOM cases

During the follow-up period per protocol, a total of 333 clinical AOM episodes were recorded in the 11Pn-PD group and 499 in the control group.

Table 3 presents the protective efficacy of the 11Pn-PD vaccine and both 7-valent vaccines previously tested in Finland (Eskola et al., N Engl J Med 2001; 344: 403-409 and Kilpi et al., Clin Infect Dis 2003 37: 1155-64) against any episode of AOM and AOM caused by different pneumococcal serotypes, H. influenzae, NTHi and M. catarrhalis.

A statistically significant and clinically relevant reduction of 33.6 % of the total burden of AOM disease was achieved with 11Pn-PD, independently of the etiology (Table 3).

The total efficacy against AOM episodes due to any of the 11 pneumococcal serotypes contained in the 11Pn-PD vaccine was 57.6% (Table 3).

Another important finding in the current study is the 35.6% protection provided by the 11Pn-PD vaccine against AOM caused by H. influenzae (and especially a 35.3% protection provided by NTHi). This finding is of major clinical significance, due to the increased importance of H. influenzae as a major cause of AOM in the pneumococcal conjugate vaccine. In line with the protection provided against AOM, the 11Pn-PD vaccine also reduced the nasopharyngeal presence of H. influenzae after the booster dose at the second year of age. These findings contrast with previous observations in Finland, where for both 7-valent pneumococcal conjugate vaccines, an increase in AOM episodes due to H. influenzae, (Eskola et al., And Kilpi ycolj as evidence of substitution) was observed. etiological

A clear correlation could not be established between the protection against the AOM episodes due to Hi and the levels of antibodies against the protein vehicle D, and the concentrations of post-primary anti-PD IgG antibodies in those vaccinated with 11Pn-PD, which were maintained Free of AOM-Hi episodes were essentially the same as post-primary PD anti-PD IgG antibody levels measured in those vaccinated with 11Pn-PD who developed at least one episode of AOM Hi during the period of efficacy monitoring. However, although the correlation between the biological impact of the vaccine and the immunogenicity for post-primary anti-PD IgG can not be established, it is reasonable to assume that the PD protein carrier, which is highly conserved among strains of H. influenzae, has contributed greatly to the induction of production against Hi.

The effect on AOM disease was accompanied by an effect on the nasopharyngeal presence that was of similar magnitude for the pneumococcal serotype vaccine and H. influenzae (Figure 1). This reduction of the nasopharyngeal presence of H. influenzae in those vaccinated with the PD conjugate supports the hypothesis of a direct protective effect of the conjugate-PD vaccine against H. influenzae, even if the protective efficacy could not be correlated with the immune responses of anti-PD IgG as measured by ELISA.

In the following example, a chinchilla otitis media model was used with pools of sera from children immunized with the 11-valent formulation of this example or with the 10-valent vaccine of Example 2 (see also Table 1 and Table 2 and Table 2). comments below) Both groupings induce a significant reduction of the percentage of animals with otitis media with respect to the grouping of pre-immune sera. There is no significant difference between the 10 and 11-valent immune clusters. This shows that both vaccines have a similar potential to induce protection against otitis media produced by a non-typable H. influenzae in this model.

Figure imgf000035_0001

Example 4:

Selection of protein vehicle for serotype 19F

ELISA test used

The ELISA method of inhibition is essentially based on a test proposed in 2001 by Concepción and Frasch and which was reported by Henckaerts et al., 2006, Clinical and Vaccine Immunology 13: 356-360. Briefly, purified pneumococcal polysaccharides were mixed with methylated human serum albumin and adsorbed on Nunc Maxisorp ™ high binding microtiter plates (Roskilde, DK) overnight at 4 ° C. Plates were blocked with 10% fetal bovine serum (FBS) in PBS for 1 hour at room temperature with shaking. The serum samples were diluted in PBS containing 10% FBS, 10 pg / ml cell wall polysaccharides (SSI) and 2 pg / ml pneumococcal polysaccharide serotype 22F (ATCC), and further diluted in the plates of microtiter with the same buffer. An internal reference calibrated against the conventional 89-SF serum was treated in each manner using serotype-specific IgG concentrations in 89-SF and was included in each plate. After washing, bound antibodies were detected using a peroxidase-conjugated anti-human IgG monoclonal antibody (Stratech Scientific Ltd., Soham, UK) diluted in 10% FBS (in PBS), and incubated for 1 hour at room temperature. environment with restless. The color was developed using a single tetramethylbenzidine component ready for use of the peroxidase enzyme immunoassay substrate kit (BioRad, Hercules, CA, US) in the dark at room temperature. The reaction was stopped with 0.18 M H2SO4, and the optical density was read at 450 nm. The serotype-specific IgG concentrations (in pg / ml) in the samples were calculated by referencing the optical density points in the defined limits with respect to the internal reference curve of the serum, which was modeled by a logistic equation of 4 parameters calculated with SoftMax Pro ™ software (Molecular Devices, Sunnyvale, CA). The cut-off point of the ELISA was 0.05 pg / ml of IgG for all serotypes, taking into account the limit of detection and the limit of quantification.

Opsono-phagocytosis test

At the WHO consultative meeting in June 2003, the use of an OPA assay was recommended as established in Romero-Steiner et al., Clin Diagn Lab Immunol 2003 10 (6): pp.1019-1024. This protocol was used to test the OPA activity of the serotypes in the following tests.

Preparation of conjugates

The studies included 11Pn-PD & Di-001 and 11Pn-PD & Di-007, three 11-valent vaccine formulations (Table 4) in which 3 pg of the 19F polysaccharide was conjugated with diphtheria toxoid (19F-DT) instead of 1 pg of polysaccharide conjugated with protein D (19F-PD). The parameters for the studies of 11Pn-PD, 11 Pn-PD & Di-001 and 11 Pn-PD & Di-007 are disclosed in Tables 5, 6 and 7, respectively.

Anti-pneumococcal antibody responses and OPA activity against 19F serotype one month after primary vaccination with these 19F-DT formulations are shown in Table 8 and 9, respectively . Table 10 shows the antibody concentrations by 22F ELISA and the percentages of subjects reaching the threshold of 0.2 pg / ml before and after the 23-valent reinforcement vaccination alone of polysaccharide.

The opsono-phagocytosis activity was clearly improved for the antibodies induced with these 19F-DT formulations as demonstrated by the higher seropositivity rates (opsono-phagocytosis titers> 1: 8) and OPA GMT one month after vaccination primary (Table 9). One month after 23-valent reinforcement vaccination with only polysaccharide, the opsono-phagocytosis activity of 19F antibodies was maintained significantly better for children sensitized with the 19F-DT formulations (Table 11).

Table 12 presents immunogenicity data after booster dose with 11Pn-PD in pre-sensitized children with pre-sensitized 19F-DT or 19F-PD conjugates compared to a 4a consecutive dose of Prevnar®. Given the cases of penetration exposed after the introduction of Prevnar® in the USA. In the US, the improvement of opsono-phagocytosis activity against the 19F serotype when conjugated with the DT protein carrier may be an advantage for candidate vaccines.

Table 13 provides the ELISA and OPA data for the 19F-DT conjugate with respect to the cross-reaction of serotype 19A. It was discovered that 19F-DT induces little OPA activity but significant against 19A.

Table 4. Conjugated pneumococcal vaccine formulations used in clinical studies.

Figure imgf000037_0001

Table 5 Specific conditions of activation / coupling / inactivation of conjugates of PS of S.

pneumoniae- Protein D / TT / DT

Figure imgf000037_0002

Figure imgf000037_0003

Table 6. Activation / coupling / inactivation conditions of Protein D conjugates of S.

pneumoniae / DT for the study of 11 Pn-PD & Di-001

Figure imgf000038_0001

Figure imgf000038_0003

Table 7. Specific activation / coupling / inactivation conditions of Protein D conjugates of S.

pneumoniae / DT for the study 11 Pn-PD & Di-007

Figure imgf000038_0002

(continuation)

Figure imgf000039_0003

Figure imgf000039_0002

Table 8. Percentage of subjects with an antibody concentration of 19F> 0.20 pg / ml and geometric mean concentration of antibodies (GMC with 95% CI, pg / ml) one month after primary vaccination with 1 pg of 19F- PD, 3 pg of 19F-DT or Prevnar (2 pg 19F-CRM) (Total cohort)

Figure imgf000039_0001

(continuation)

Figure imgf000040_0001

Table 9. Percentage of subjects with an OPA titre 19F> 0.20 mg / ml and geometric mean of 19F antibody concentrations (GMC with 95% CI, mg / ml) one month after primary vaccination with 1 | jg of 19F-PD, 3 | jg of 19F-DT or Prevnar (2 | jg 19F-CRM) (Total Cohort)

Figure imgf000040_0002

Table 10. Percentage of subjects with an antibody concentration of 19F> 0.20 pg / ml and GMC of 19F antibody (pg / ml) before and one month after 23-valent reinforcement with only polysaccharide in children sensitized with 1 pg of 19F -PD, 3 pg of 19F-DT or Prevnar (2 pg 19F-CRM) (Total cohort)

Figure imgf000040_0003

Table 11. Percentage of subjects with an OPA titre 19F> 1: 8 and GMT of OPA 19F before and at least following the 23-valent booster alone with polysaccharide in children sensitized with 1 pg of 19F-PD, 3 pg of 19F- DT or Prevnar (2 pg 19F-CRM) (Total Cohort)

Figure imgf000041_0001

Table 12. Percentage of subjects with antibody concentrations> 0.2 pg / ml, OPA> 1: 8 and GMC / GMT against 19F pneumococci one month after reinforcement with 11Pn-PD or Prevnar in children sensitized with 1 pg of 19F- PD, 3 pg of 19F-DT or Prevnar (2 pg 19F-CRM) (Total cohort)

Figure imgf000041_0002

Table 13. Percentage of subjects with antibody concentrations> 0.2 pg / ml, OPA> 1: 8 and GMC / GMT against 19A pneumococci one month after primary vaccination with 1 pg of 19F-PD, 3 pg of 19F- DT or Prevnar (2 pg 19F-CRM) (Total Cohort)

Figure imgf000041_0003

Example 5: Adjuvant experiments in preclinical models: impact on the immunogenicity of 11-valent polysaccharide conjugates in elderly Rhesus monkeys

To optimize the response triggered by pneumococcal conjugate vaccines in the elderly population, GSK formulated a vaccine with 11-valent conjugated polysaccharides (PS) with a new adjuvant, adjuvant C -see later.

Immunized intramuscularly (IM) groups of elderly Rhesus monkeys (14 to 28 years old) on days 0 and 28 with 500 | jl of 11-valent PS conjugates adsorbed on 315 | jg of AIPO4 or conjugates of PS 11- valent mixed with Adjuvant C.

In both vaccine formulations, the 11-valent PS conjugates were composed of the following conjugates PS1-PD, PS3-PD, PS4-PD, PS5-PD, PS7F-PD, PS9V-PD, PS14-PD, PS18C-PD, PS19F-PD, PS23F-DT and PS6B-DT. The vaccine used was 1/5 the dose of the human dose of the vaccine (5 jg of each saccharide per dose for humans except 6B [10 jg]) conjugated according to the conditions of Table 6 (Example 4) , except for 19F that was made according to the following CDAP procedure conditions: the saccharide with size modification at 9 mg / ml, PD at 5 mg / ml, an initial PD / PS ratio of 1.2 / 1, a concentration of CDAP of 0.75 mg / mg PS, pHa = pHc = pH 9.0 / 9.0 / 9.0 and a coupling time of 60 min.

ELISA levels of anti-PS IgG and opsono-phagocytosis titres were measured in the serum collected on day 42. The frequency of anti-PS3 memory B lymphocytes was measured by Elispot from peripheral blood cells collected on the day 42

According to the results shown hereinafter, Adjuvant C improved the immunogenicity of 11-valent PS conjugates with AIPO4 in elderly monkeys. The new adjuvant increased the IgG responses to PS (Figure 1) and the antibody titers opsono-phagocytosis (Table 14). There was also evidence to support that the frequency of PS3-specific memory B lymphocytes was increased with the use of Adjuvant C (Figure 2).

Figure imgf000043_0001

B lymphocyte elispot

The principle of the assay is based on the fact that B lymphocytes mature to plasma cells in vitro after culture with CpG for 5 days. The antigen-specific plasma cells generated in vitro can be easily detected and are therefore enumerated using the Elispot B lymphocyte assay. The number of plasma cells reflects the frequency of memory B cells at the beginning of the culture.

Briefly, cells generated in vitro are incubated in culture plates coated with antigen. The antigen-specific plasma cells form antigen / antibody spots, which are detected by a conventional immunoenzymatic method and counted as memory B cells.

In the present study, the polysaccharides were used to coat the culture plates in order to count the respective memory B lymphocytes. The results are expressed as a frequency of PS memory-specific B lymphocytes per million B memory lymphocytes.

The study shows that Adjuvant C may be able to alleviate the problem of PS3 booster (see 5th International Symposium on Pneumococcal and Pneumococcal Diseases, 2-6 April 2006, Alice Springs, Central Australia. serotype 3 pneumococcal conjugate, Schuerman L, Prymula R, Poolman J. Abstract book p 245, PO10.06).

Example 6. Efficacy of detoxified pneumolysin (dPly) as a vehicle for the immunogenicity of PS 19F in young Balb / c mice

Groups of 40 females of Balb / c mice (4 weeks old) IM were immunized on days 0, 14 and 28 with 50 μl of only 4-valent PS or PS conjugated with 4-valent dPly, both mixed with adjuvant C.

Both vaccine formulations consisted of 0.1 pg (amount of saccharide) from each of the following PS: PS8, PS12F, PS19F and PS22F.

ELISA levels of anti-PS IgG were measured in sera collected on day 42.

The anti-PS19F response, shown as an example in Figure 3, was strongly increased in the mice given the 4-valent dPly conjugates compared to the mice immunized with the PS alone. The same improvement was observed with anti-PS8, 12F and 22F IgG responses (data not shown).

Example 7. Efficacy of Protein D of the pneumococcal Histidine triad (PhtD) as a protein carrier to increase the immunogenicity of PS 22F in young Balb / c mice

Groups of 40 females of Balb / c mice (4 weeks old) IM were immunized on days 0, 14, and 28 with 50 μl of only 4-valent PS or PS conjugated with 4-valent PhtD, both mixed with Adjuvant C .

Both vaccine formulations consisted of 0.1 pg (amount of saccharide) from each of the following PS: PS8, PS12F, PS19F and PS22F.

ELISA levels of anti-PS IgG were measured in the sera collected on day 42.

The anti-PS22F response, shown as an example in Figure 4, was strongly increased in the mice given the 4-valent PhtD conjugates compared to the mice immunized only with the PS. The same improvement was observed for anti-PS8, 12F and 19F IgG responses (data not shown).

Example 8. Immunogenicity in elderly C57Bl mice of 13-valent PS conjugates containing 19A-dPly and 22F-PhtD

Groups of 30 old C57Bl mice (> 69 weeks of age) IM were immunized on days 0, 14 and 28 with 50 μl of 11-valent PS conjugates or 13-valent PS conjugates, both mixed with Adjuvant C (see below) .

The 11-valent vaccine formulation was composed of 0.1 pg of saccharides from each of the following conjugates: PS1-PD, PS3-PD, PS4-PD, PS5-PD, PS6B-PD, PS7F-PD, PS9V-PD , PS14-PD, PS18C-TT, PS19F-DT and PS23F-PD (see Table 1 and the commentary on the 11-valent vaccine treated under Table 2). The 13-valent vaccine formulation further contained 0.1 pg of conjugates PS19A-dPly and PS22F-PhtD (see Table 1 and the comment of the 13-valent vaccine under Table 2 [using 22F directly conjugated]). In group 2 and 4 the pneumolysin vehicle was detoxicized with a GMBS treatment, in group 3 and 5 it was made with formaldehyde. In groups 2 and 3 PhtD was used to conjugate PS22F, in groups 4 and 5 a fusion of PhtD-E (the VP147 construction of WO 03/054007) was used. In group 6, 19A was conjugated with diphtheria toxoid and 22F with protein D.

ELISA levels of anti-PS19A and 22F IgG were measured in individual sera collected on day 42. The IgG ELISA response generated by the other PSs was measured in pooled serum.

19A-dPly and 22F-PhtD administered in the 13-valent conjugate vaccine formulation demonstrated that they were immunogenic in old C57Bl mice (Table 15). The immune response induced against other PSs was not negatively impacted in mice given the 13-valent formulation compared to those immunized with the 11-valent formulation.

Table 15. Immunogenicity of PS in old C57BI mice (post-III IgG levels)

Figure imgf000045_0001

Example 9. Immunogenicity in young Balb / c mice of 13-valent PS conjugates containing 19A-dPly and 22F-PhtD

Groups of 30 young Balb / c mice (4 weeks old) IM were immunized on days 0, 14 and 28 with 50 μl of 11-valent PS conjugates or 13-valent SP conjugates, both mixed with Adjuvant C ( see later).

The 11-valent vaccine formulation was composed of 0.1 g of saccharide from each of the following conjugates: PS1-PD, PS3-PD, PS4-PD, PS5-PD, PS6B-PD, PS7F-PD, PS9V-PD , PS14-PD, PS18C-TT, PS19F-DT and PS23F-PD (see Table 1 and the commentary on the 11-valent vaccine discussed under Table 2). The 13-valent vaccine formulation further contained 0.1 jg of conjugates PS19A-dPly and PS22F-PhtD (see Table 1 and comment of the 13-valent vaccine discussed below in Table 2 [using 22F directly conjugated]). In group 2 and 4 the pneumolysin vehicle was detoxicized with a GMBS treatment, in group 3 and 5 it was made with formaldehyde. In groups 2 and 3, PhtD was used to conjugate the PS 22F. In Groups 4 and 5, a PhtD_E fusion (the VP147 construction of WO 03/054007) was used. In group 6, 19A was conjugated with diphtheria toxoid and 22F with protein D.

Anti-PS19A IgG ELISA levels were measured in individual sera collected on day 42. The IgG ELISA response generated against the other PSs was measured in pooled sera.

The 19A-dPly and 22F-PhtD administered in the 13-valent conjugated vaccine formulation proved to be immunogenic in young Balb / c mice (Table 16). The immune response induced against the other PSs was not negatively impacted in mice to which the 13-valent formulation was administered compared to those immunized with the 11-valent formulation.

Table 16. Immunogenicity of PS in young Balb / c mice (post-III IgG levels)

Figure imgf000046_0001

(continuation)

Figure imgf000047_0001

Example 10. Immunogenicity in guinea pigs of 13-valent PS conjugates containing 19A-dPly and 22F-PhtD

Groups of 20 young guinea pigs (Cepa Hartley, 5 weeks old) IM were immunized on days 0, 14 and 28 with 125 | j | of conjugates of PS 11-valent or conjugates of PS 13-valent, both mixed with Adjuvant C (see below).

The 11-valent vaccine formulation was composed of 0.25 jg of saccharide from each of the following conjugates: PS1-PD, PS3-PD, PS4-PD, PS5-PD, PS6B-PD, PS7F-PD, PS9V-PD , PS14-PD, PS18C-TT, PS19F-DT and PS23F-PD (see Table 1 and the commentary on the 11-valent vaccine discussed below in Table 2). The 13-valent vaccine formulation further contained 0.1 jg of conjugates PS19A-dPly and PS22F-PhtD (see Table 1 and comment dealing with the 13-valent vaccine below in Table 2 [using 22F directly conjugated]). In group 2 and 4 the pneumolysin vehicle was detoxicized with a GMBS treatment, in group 3 and 5 it was made with formaldehyde. In groups 2 and 3, PhtD was used to conjugate the PS 22F. In Groups 4 and 5, a PhtD_E fusion (the VP147 construction of WO 03/054007) was used. In group 6, 19A was conjugated with diphtheria toxoid and 22F with protein D.

Anti-PS19A IgG ELISA levels were measured in individual sera collected on day 42. The IgG ELISA response generated against the other PSs was measured in pooled sera.

Table 17. Immunogenicity in young guinea pigs (post-III IgG levels)

Figure imgf000048_0001

Example 11: Formulations to be made and tested

The following formulations were made (using the 13-valent vaccine of Table 1 and serotype 3 of Table 5 - see the comment that is about the 14-valent vaccine below in Table 2 [using 22F directly conjugated or by means of of an ADH linkage]). The saccharides were formulated with aluminum phosphate and 3D-MPL as shown below.

Figure imgf000049_0001

b) The same saccharide formulation was adjuvanted with each of the following adjuvants: - The concentration of the component emulsion per 500 ul is shown in the table below.

Figure imgf000049_0002

Adjuvant A4 Adjuvant A5 Adjuvant A6 Adjuvant A7

Ingredients Emulsion Emulsion Emulsion Emulsion

250 | jl ac / ag 250 j l ac / g 125 j l ac / ag 50 j l ac / ag

Alpha Tocopherol 11.88 mg 11.88 mg 5.94 mg 2.38 mg

Squalene 10.7 mg 10.7 mg 5.35 mg 2.14 mg

Tween 80 4.85 mg 4.85 mg 2.43 mg 0.97 mg

3D-MPL 50 jg 25 jg 25 jg 10 jg

c) The saccharides were also formulated with two adjuvants based on liposomes:

Composition of Adjuvant B1

Qualitative Quantitative (per dose of 0.5 ml)

Liposomes:

- DOPC1 mg

- cholesterol 0.25 mg

3D-MPL 50 jg

QS21 50 jg

Buffer KH 2 PO 4 A 12 4 mg

Buffer Na2HPO4-i0.290 mg

NaCl 2.922 mg

(100 mM)

Solvent WFI q.s. up to 0.5 ml

pH 6.1

1. Total concentration PO 4 = 50 mM

Composition of Adjuvant B2

Qualitative Quantitative (per dose of 0.5 ml)

Liposomes:

- DOPC1 mg

- cholesterol 0.125 mg

3D-MPL 25 jg

QS21 25 jg

Buffer KH 2 PO 4 A 12 4 mg

Buffer Na2HPO4-i0.290 mg

NaCl 2.922 mg

(100 mM)

Solvent WFI q.s. up to 0.5 ml

pH 6.1

d) The saccharides were also formulated with Adjuvant C (see above for other compositions that have been used with this adjuvant):

Qualitative Quantitative (per dose of 0.5 ml)

Oil in water emulsion: 50 μl

- squalene 2,136 mg

- a-tocopherol 2,372 mg

- Tween 800.97 mg

- Cholesterol 0.1 mg

3D-MPL 50 jg

QS21 50 jg

Buffer Kh 2P04-i0,470 mg

Buffer Na2HPO4-i0.219 mg

NaCl 4,003 mg

(137 mM)

KCl 0.101 mg

(2.7 mM)

Solvent WFI q.s. ad 0.5 ml

pH 6.8

Example 12. Impact of chemical conjugation on the immunogenicity of the 22F-PhtD conjugate in Balb / c mice

Groups of 30 females of Balb / c mice were immunized intramuscularly (IM) on days 0, 14 and 28 with 13-valent PS formulations containing PS 1, 3, 4, 5, 6B, 7F, 9V, 14 , 18C, 19A, 19F, 22F and 23f (dose: 0.3 | jg saccharide / conjugate for PS 4, 18C, 19A, 19F and 22F and 0.1 | jg saccharide / conjugate for the other PS).

PS 18C was conjugated with tetanus toxoid, 19F with diphtheria toxoid, 19A with Ply detoxified with formaldehyde, 22F with PhtD and the other PS with PD.

Two formulations, consisting of 22F-PhtD prepared by direct CDAP chemistry or 22F-AH-PhtD (PS derived with ADH), were compared. see Example 2, Table 1 and the commentary under Table 2 for the characteristics of the 13-valent vaccine made or with 22F conjugated directly or by means of an ADH spacer. The vaccine formulations were supplemented with adjuvant C.

ELISA levels of anti-PS22F IgG and opsono-phagocytosis titres were measured in the sera collected on day 42.

22F-AH-PhtD proved to be much more immunogenic than 22F-PhtD in terms of both IgG levels (Figure 5) and opsono-phagocytosis titers (Figure 6).

Example 13. Impact of the new adjuvants on the immunogenicity of conjugated PS of Streptococcus pneumoniae

Groups of 40 female Balb / c mice were immunized IM on days 0, 14 and 28 with 13-valent PS formulations containing PS 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A , 19F, 22F and 23F (dose: 0.3 jg / conjugate for PS 4, 18C, 19A, 19F and 22F and 0.1 jg / conjugate for the other PS).

PS 18C was conjugated with Tetanus Toxoid, 19F with Diphtheria Toxoid, 19A with Ply detoxicated with formaldehyde, 22F with PhtD and the other PS with PD. See Example 2, Table 1 and the comment under Table 2 for the characteristics of the 13-valent vaccine made with 22F conjugated directly.

Four formulations, supplemented with AIPO4, adjuvant A1, adjuvant A4 or adjuvant A5 were compared.

The levels of IgG ELISA anti-PS, Ply, PhtD and PD were measured in the sera collected on day 42 and grouped by group.

The following ratio was calculated for each antigen: IgG level induced with the new adjuvant tested / level of IgG induced with AIPO4.

All the new adjuvants tested improved at least 2-fold the immune responses to the 13-valent conjugates compared to the classical AIPO4 formulation (Figure 7).

Example 14. Protective efficacy of a detoxified PhtD / Ply combination in a mono pneumococcal pneumonia model

Groups of 6 Rhesus monkeys (3 to 8 years of age), which were selected from those with the lowest levels of pre-existing anti-19F antibodies, were immunized intramuscularly on days 0 and 28 with conjugates of PS 11- valent (ie, 1 jg of PS 1, 3, 5, 6B, 7F, 9V, 14 and 23F, and 3 jg of PS 4, 18C and 19F [of saccharide] or PhtD (10 jg) Ply detoxified with formaldehyde ( 10 jg) or the adjuvant alone.

PS 18C was conjugated with tetanus toxoid, 19F with diphtheria toxoid and the other PS with PD. See Example 2, Table 1 and comment under Table 2 for characteristics of the 11-valent vaccine. All formulations were supplemented with adjuvant C.

Type 19F pneumococci (5,108 cfu) were inoculated into the right lung on day 42. The colonies were counted in bronchoalveolar lavages collected on days 1, 3 and 7 post-challenge. The results were expressed as the number of animals per group killed, with the lung colonized or free on day 7 after the challenge.

As shown in Figure 8, good protection was obtained very close to statistical significance (despite the low number of animals used) with the 11-valent conjugates and the PhtD dPly combination (p <0.12, test Fisher's exact) compared to the adjuvant group alone.

Claims (13)

  1. An immunogenic composition comprising conjugates of S. pneumoniae capsular saccharides of serotypes 19A and 19F in which 19A is conjugated with a first bacterial toxoid that is pneumolysin, diphtheria toxoid or CRM197 and 19F is conjugated with a second bacterial toxoid that is diphtheria toxoid or CRM197 and further comprising conjugates of the capsular saccharides of S. pneumoniae 4, 6B, 9V, 14, 18C, 23F, 1, 5 and 7F, in which the average size of saccharide 19A is between 110 and 700 kDa
  2. 2. The immunogenic composition of claim 1, further comprising a conjugate of the 22F capsular saccharide of S. pneumoniae.
  3. 3. The immunogenic composition of any of the preceding claims, wherein the average size of saccharide 19A is between 110-300, 120-200, 130-180 or 140-160 kDa.
  4. 4. The immunogenic composition of any preceding claim, wherein the saccharide 19A has been dimensioned by a factor of not more than x5.
  5. 5. The immunogenic composition of any preceding claim, wherein the saccharide 19A has been sized by microfluidization.
  6. 6. The immunogenic composition of any preceding claim, wherein the average size of the saccharides is above 50 kDa.
  7. The immunogenic composition according to claim 6, comprising serotype 1 having an average saccharide size of between 300 and 400 kDa.
  8. 8. The immunogenic composition according to claim 6 and 7, comprising serotype 5 having an average saccharide size of between 350 and 450 kDa.
  9. 9. The immunogenic composition according to any of claims 6 to 8, comprising serotype 7F having an average saccharide size of between 200 and 300 kDa.
  10. 10. A vaccine comprising the immunogenic composition of any one of claims 1 to 9 and a pharmaceutically acceptable excipient.
  11. 11. A method of manufacturing a vaccine according to claim 10, comprising the step of mixing the immunogenic composition of any of claims 1 to 9 with a pharmaceutically acceptable excipient.
  12. 12. The immunogenic composition of claims 1-9 or the vaccine of claim 10, for use in the treatment or prevention of disease caused by Streptococcus pneumoniae infection .
  13. The immunogenic composition of claims 1-9 or the vaccine of claim 10, for use in the treatment or prevention of disease caused by Streptococcus pneumoniae infection according to claim 12, wherein the disease is either or both of pneumonia or invasive pneumococcal disease (IPD) of elderly humans, exacerbations of chronic obstructive pulmonary disease (COPD) of elderly humans, otitis media of lactating humans, meningitis and / or bacteremia of lactating humans, or pneumonia and / or conjunctivitis of lactating human beings.
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GB0620337A GB0620337D0 (en) 2006-10-12 2006-10-12 Vaccine
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ES15195398T Active ES2707499T3 (en) 2005-12-13 2006-12-20 Pneumococcal polysaccharide conjugate vaccine
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